2-{[N-(2-amino-3-(heteroaryl or aryl) propionyl)-aminoacyl]-amino}-alkylboronic acid derivatives

ABSTRACT

The present invention relates to compounds of formula I  
                 
wherein the substituents are as defined in the description, to processes for the preparation thereof, to pharmaceutical preparations comprising those compounds, and to the use thereof in the preparation of pharmaceutical compositions for the therapeutic treatment of warm-blooded animals, including humans.

The invention relates to 2-{[N-(2-amino-3-(heteroaryl or aryl)propionyl)-aminoacyl]-amino}-alkylboronic acid derivatives, to processes for the preparation thereof, to pharmaceutical preparations comprising those compounds, and to the use thereof in the preparation of pharmaceutical compositions for the therapeutic treatment of warm-blooded animals, including humans.

The invention relates to compounds of formula I

wherein

-   -   R₁ is unsubstituted or substituted aryl; arylalkylcarbonyl,         wherein the aryl moiety is unsubstituted or substituted;         unsubstituted or substituted heterocyclyl; or         heterocyclylalkylcarbonyl, wherein the heterocyclyl moiety is         unsubstituted or substituted;     -   R₂ is unsubstituted or substituted aryl or unsubstituted or         substituted heteroaryl;     -   R₃ is hydrogen, unsubstitued or substituted aryl or alkyl which         is unsubstituted or substituted by         -   unsubstituted or substituted cycloalkyl,         -   unsubstituted or substituted aryl, or         -   unsubstituted or substituted heteroaryl comprising at least             one nitrogen atom;     -   R₄ is a moiety of the formula IA,         wherein A₁ and A₂ are hydroxy or substituted hydroxy, or         together with the binding boron atom and the two binding oxygen         atoms form a ring of the formula IA*,         wherein W is alkylene, substituted alkylene, unsubstituted or         substituted cycloalkylene, unsubstituted or substituted         bicycloalkylene or unsubstituted or substituted         tricycloalkylene;     -   and R₅ is unsubstituted or substituted alkyl, unsubstituted or         substituted aryl, unsubstituted or substituted heterocyclyl, or         unsubstituted or substituted cycloalkyl;     -   or salts thereof.

Within the context of the present disclosure the general terms used hereinbefore and hereinafter preferably have the following meanings:

Aryl preferably has a ring system of not more than 20 carbon atoms, especially not more than 12 carbon atoms, is preferably mono-, bi- or tric-cyclic, and is unsubstituted or substituted, preferably in each case unsubstituted or substituted phenyl or (especially 1- or 2-)naphthyl, one or more substituents preferably being independently selected from the group consisting of an aliphatic radical; free, etherified or esterified hydroxy; free or esterified carboxy; formyl; alkanoyl; unsubstituted, mono- or di-substituted amino; mercapto; sulfo; alkyl-thio; carbamoyl; N-alkyl-carbamoyl; N,N-di-alkyl-carbamoyl; phenyl; naphthyl; heterocyclyl, especially pyridyl; cyano and nitro, more preferably being selected from alkyl, e.g. methyl, ethyl or propyl; alkoxy, e.g. methoxy or ethoxy; di-substituted amino, e.g. dimethylamino; halogen, e.g. chloro or bromo; halogen-alkyl, e.g. trifluoromethyl; and phenyl, (especially 1- or 2-)-naphthyl, and heterocyclyl, especially as defined below, especially pyridyl, e.g. 3-, 4- or especially 2-pyridyl, each of which is unsubstituted or substituted with one or more, especially up to three, substituents, especially independently selected from the other aryl substitutents just mentioned. Aryl R₁ is more preferably biphenylyl, especially 2-, 4- or preferably 3-biphenylyl, pyridylphenyl, especially 4-, 3- or most especially 2-pyridyl-(2-, 4- or preferably 3-)phenyl, or lower alkyl-phenyl, especially propyl-phenyl, such as 2-, 4- or especially 3-isopropylphenyl. Arylalkylcarbonyl R₁ (with unsubstituted or preferably substituted aryl) is preferably aryl-lower alkylcarbonyl with aryl as defined above, more preferably phenyl-lower alkyloxy-phenyl-lower alkylcarbonyl, especially 2-, 4- or preferably 3-benzyloxy-phenyl-acetyl or -propionyl, pyridyl-lower alkyloxyphenyl-lower alkylcarbonyl, especially 2-, 4- or preferably 3-(pyridin-2-, 4- or preferably -3-)-acetyl or -propionyl, or phenyl-lower alkylcarbonyl, especially phenyl-2- or preferably 3-phenyl-propionyl or phenylacetyl, wherein phenyl is unsubstituted or substituted by up to three substitutents independently selected from lower alkoxy, especially methoxy, halogen, especially fluoro or chloro, or halogen-lower alkyl, such as trifluoromethyl. Unsubstituted or substituted aryl R₂ or (independently) R₃ is preferably mono-, di- or trisubstituted phenyl, especially substituted by up to four substituents independently selected from the substitutents mentioned for aryl, especially from hydroxy, lower alkoxy (most preferred), preferably methoxy, halogen, preferably fluoro or chloro, and halogen-lower alkyl, preferably trifluoromethyl, especially phenyl substituted by up to three lower alkoxy, preferably methoxy, substituents, or in case of R₃ unsubstituted phenyl, or further unsubstituted or substituted napthyl, especially 1- or 2-naphthyl that is unsubstituted or substituted by up to four substituents independently selected from the substitutents mentioned for aryl, especially from hydroxy, lower alkoxy (most preferred), preferably methoxy, halogen, preferably fluoro or chloro, and halogen-lower alkyl, preferably trifluoromethyl.

Unsubstituted heterocyclyl is preferably a heterocyclic radical that is unsaturated, saturated or partially saturated in the bonding ring and is preferably monocyclic or in a broader sense bicyclic or tricyclic ring; has 3 to 24, more preferably 4 to 16 ring atoms; wherein at least in the ring bonding to the radical of the molecule of formula I one or more, preferably one to four, especially one or two carbon atoms of a corresponding aryl radical are substituted by a heteroatom selected from the group consisting of nitrogen, oxygen and sulfur, the bonding ring preferably having 4 to 12, especially 5 to 7 ring atoms; heteroaryl being unsubstituted or substituted by one or more, especially 1 to 3, substitutents independently selected from the group consisting of the substituents defined above as substituents of substituted aryl; and especially being a heteroaryl radical selected from the group consisting of imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazolidinyl, pyranyol, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoqionolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, benzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, isochromanyl and chromanyl, each of these radicals being unsubstituted or substituted by one to two radicals selected from the group consisting of lower alkyl, especially methyl or tert-butyl, lower alkoxy, especially methoxy, and halo, especially bromo or chloro; pyridyl, especially 2- or 3-pyridyl, or indolyl is especially preferred, in a broader aspect lower alkylpyridyl, pyrimidinyl or lower alkylpyrimidinyl, halo-lower alkylpyridyl, lower alkoxy-pyridyl, di-lower alkyl-pyridyl, or halo-pyridyl. Heterocyclyl is unsubstituted or substituted by one or more, preferably up to three, substitutents independently selected from those mentioned above for aryl (where heterocyclyl as substituent of heterocyclyl carries no further heterocyclyl substituent other than pyridyl or indolyl) and from aryl as defined above, especially phenyl, especially those mentioned as being preferred. Unsubstituted heterocyclyl is preferred.

In heterocyclylalkylcarbonyl R₁, the heterocyclyl moiety is preferably substituted or especially unsubstituted heterocyclyl as mentioned above; preferred is substituted or preferably unsubstituted heterocyclyl-lower alkyl, especially with terminal substituted or preferably unsubstituted heterocyclyl, with heterocyclyl as described above; preferred is pyridyl-lower alkylcarbonyl, such as -acetyl or -propionyl.

As R₁, unsubstituted or substituted aryl or substituted aryl-lower alkylcarbonyl is preferred over the other meanings.

Heteroaryl R₂ is preferably unsubstituted or substituted heteroaryl as mentioned above, especially indolyl that is unsubstituted or substituted by one or more, especially up to three, substitutents independently selected from those mentioned above for substituted aryl, especially from hydroxy, lower alkoxy (most preferred), preferably methoxy, halogen, preferably fluoro or chloro, and halogen-lower alkyl, preferably trifluoromethyl.

R₂ is preferably substituted aryl.

An aliphatic radical preferably has up to 12 carbon atoms, preferably up to 7 carbon atoms, most preferably up to 4 carbon atoms, and is an aliphatic hydrocarbon radical, that is to say such an unsubstituted or substituted alkynyl, alkenyl or preferably alkyl radical, more preferably lower alkyl, especially methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl or tert-butyl.

Alkyl, which may be branched or linear, preferably has up to 12 carbon atoms, and is more preferably lower alkyl. Alkyl R₃ is preferably lower alkyl, especially isobutyl.

The prefix “lower” denotes a radical having up to and including 7, preferably up to and including 4, carbon atoms.

Lower alkyl is, preferably, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl or n-heptyl, preferably isobutyl, sec-butyl, tert-butyl, isopropyl, ethyl or methyl, most preferably isopropyl, ethyl or methyl.

Etherified hydroxy is, for example, alkoxy, especially lower alkoxy, such as ethoxy or methoxy, aryloxy, especially phenyloxy, aryl-lower alkoxy, especially phenyl-lower alkoxy, heterocyclyloxy, especially pyridyloxy, or heterocyclyl-lower alkoxy, especially pyridyl-lower alkoxy (aryl and heterocyclyl preferably have the meanings given above).

Esterified hydroxy is preferably hydroxy esterified by an organic carboxylic acid, such as an alkanoic acid, for example lower alkanoyloxy.

Esterified carboxy is, for example, alkoxycarbonyl, especially lower alkoxycarbonyl, such as e.g. methoxycarbonyl.

Mono- or di-substituted amino is, preferably, N-alkylamino or N,N-dialkylamino, especially N-lower alkylamino or lower N,N-di-lower alkylamino, such as N-methylamino or N,N-dimethylamino.

Halogen is fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

Unsubstituted or substituted cycloalkyl preferably has up to 12, more preferably 3 to 8 ring carbonyl atoms and is substituted by one or more, especially up to three, substitutents independently selected from those mentioned for substituted aryl, or preferably unsubstituted. Preferred is cyclopentyl, cyclohexyl or cycloheptyl.

In alkyl R₃ substituted with unsubstituted or substituted cycloalkyl, alkyl is preferably as defined above, more preferably lower alkyl, especially isopropyl, and is (preferably terminally) substituted by cycloalkyl as defined above.

In alkyl R₃ substituted with unsubstituted or substituted aryl, alkyl is preferably as defined in the last paragraph, and aryl is defined as above and is substituted by one or more, especially up to three, substitutents independently selected from those mentioned for substituted aryl, or unsubstituted; especially aryl is phenyl substituted by one or more, especially up to three, substitutents independently selected from halogen, especially fluoro, hydroxy or lower alkoxy, especially methoxy, or it is unsubstituted phenyl.

In alkyl R₃ substituted with unsubstituted or substituted heterocyclyl, alkyl is preferably as defined for alkyl R₃ substituted with cycloalkyl, and heterocyclyl is defined as above and is substituted by one or more, especially up to three, substitutents independently selected from those mentioned for substituted heterocyclyl, or unsubstituted.

If A₁ and A₂ each are substituted hydroxy, then substituted hydroxy is preferably alkyloxy, especially lower alkyloxy, aryloxy, especially with unsubstituted or substituted aryl as defined above, or cycloalkyloxy with unsubstituted or substituted cycloalkyl as defined above.

If A₁ and A₂ together with the binding boron atom and oxygen atoms form a ring or the formula IA* shown above, then W preferably carries the two oxygen atoms bound to the boron atom on two different carbon atoms that are spatially nearby or neighbouring carbon atoms, especially in vicinal (“1,2-”) or in “1,3”-position (relatively to each other).

Alkylene is preferably an unbranched C₂-C₁₂, preferably C₂-C₇alkylene moiety, e.g. ethylene, or propylene, in a broader aspect butylene, pentylene or hexylene, bound via two different carbon atoms as just described, preferably vicinal or in “1,3”-position. One or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or preferably N (carrying the required number of H atoms, respectively), for example in 1,5(3-aza-pentylene).

Substituted alkylene is preferably an unbranched lower alkylene moiety as defined above which is subsituted or unsubstituted by one or more, especially up to three, substituents preferably independently selected from lower alkyl, such as methyl or ethyl, e.g. in 1-methylethylene, 1,2-dimethylethylene, hydroxy, e.g. in 2-hydroxy-propylene, or hydroxy-lower alkyl, such as hydroxymethyl, e.g. in 1-hydroxymethyl-ethylene.

Unsubstituted or substituted cycloalkylene is preferably C₃-C₁₂-, more preferably C₃-C₈-cycloalkylene bound via two different carbon atoms as described for W, preferably vicinal or in “1,3”-position, such as cyclohexylene or cyclopentylene, in which one or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or N (carrying the required number of H atoms, respectively), for example in tetrahydrofurylene or tetrahydropyranylene, and may be unsubstituted or substituted by one or more, especially up to three substituents independently selected from lower alkyl, such as methyl or ethyl, hydroxy, hydroxy-lower alkyl, such as methoxy, or mono- or oligosaccharidyl bound via an oxyygen atom (“oligosaccharidyl” preferably comprising up to five saccaridyl moieties).

Unsubstituted or substituted Bicycloalkylene is preferably C₅-C₁₂-bicycloalkylene bound via two different carbon atoms as described for W, preferably vicinal or in “1,3”-position, in which one or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or N (carrying the required number of H atoms, respectively), and may be unsubstituted or substituted by one or more, especially up to three substituents independently selected from lower alkyl, such as methyl or ethyl, hydroxy and hydroxy-lower alkyl, such as methoxy. Preferred is pinanylene (2,3-(2,6,6-trimethyl-bicylo[3.1.1]heptane).

Unsubstituted or substituted tricycloalkylene is preferably C₈-C₁₂-tricycloalkylene bound via two different carbon atoms as described for W, preferably vicinal or in “1,3”-position, in which one or more, especially one, of the carbon atoms not bound to the oxygen atoms binding to the boron atom may be replaced by a heteroatom selected from O, S or N (carrying the required number of H atoms, respectively), and may be unsubstituted or substituted by one or more, especially up to three substituents independently selected from lower alkyl, such as methyl or ethyl, hydroxy and hydroxy-lower alkyl, such as methoxy.

Most preferably, R₄ is —B(OH)₂ or 2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl, especially (1S, 2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl,

In unsubstituted or substituted alkyl R₅, alkyl, which may be branched or linear, preferably has up to 12 carbon atoms, and is more preferably lower alkyl. Alkyl R₅ is preferably lower alkyl, especially isopropyl. Substituents, of which one or more, especially up to two, may be present, are independently selected from unsubstituted or substituted aryl (especially phenyl or hydroxyphenyl), unsubsituted or substituted heterocyclyl (especially imidazolyl or indolyl), unsubstituted or substituted cycloalkyl, each as defined above; hydroxy (preferred), carboxy (preferred), carbamoyl, mercapto, lower alkylthio, e.g. methylthio, phenyl, hydroxyphenyl, indolyl, imidazolyl, amino, tri-lower alkylamino, e.g. trimethylamino, lower alkanoylamino, e.g. acetylamino, guanidino, N-lower alkylguanidino, e.g. N-methylguanidino, or any other substituent completing an amino acid comprising R₅. Preferably, R₅ may be methyl, isopropyl, isobutyl, sec-butyl, mercaptomethyl, 2-methylthioethyl, phenylmethyl, hydroxyphenylmethyl, indol-3-ylmethyl, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, carbamoylmethyl, 2-carbamoylethyl, 4-aminobutyl, 3-guanidinopropyl, 5-imidazolylmethyl, carboxymethyl or 2-carboxyethyl.

Asymmetric carbon atoms of a compound of formula I that are present may exist in the (R), (S) or (R,S) configuration, preferably in the (R) or (S) configuration, most preferably in the configuration indicated in formula I* below. Substituents at a double bond or a ring may be present in cis-(=Z-) or trans (=E-) form. The compounds may thus be present as mixtures of isomers or preferably as pure isomers.

Salt-forming groups in a compound of formula I are groups or radicals having basic or acidic properties. Compounds having at least one basic group or at least one basic radical, for example amino, a secondary amino group not forming a peptide bond or a pyridyl radical, may form acid addition salts, for example with inorganic acids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono- or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids, such as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxy-benzoic acid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such as nicotinic acid or isonicotinic acid, aliphatic sulfonic acids, such as methane-, ethane- or 2-hydroxyethanesulfonic acid, or aromatic sulfonic acids, for example benzene-, p-toluene- or naphthalene-2-sulfonic acid. When several basic groups are present mono- or poly-acid addition salts may be formed.

Compounds of formula I having acidic groups, for example a free boronic acid group (—B(OH)₂, that is, in formula IA* A₁ and A₂ each are hydroxy) or a carboxy group, may form metal or ammonium salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or suitable organic amines, such as tertiary monoamines, for example triethylamine or tri-(2-hydroxy-ethyl)-amine, or heterocyclic bases, for example N-ethyl-piperidine or N,N′-dimethylpiperazine. Mixtures of saltsa are possible.

Compounds of formula I having both acidic and basic groups can form internal salts.

For the purposes of isolation or purification, as well as in the case of compounds that are used further as intermediates, it is also possible to use pharmaceutically unacceptable salts, e.g. the picrates. Only pharmaceutically acceptable, non-toxic salts may be used for therapeutic purposes, however, and those salts are therefore preferred.

Owing to the close relationship between the novel compounds in free form and in the form of their salts, including those salts that can be used as intermediates, for example in the purification of the novel compounds or for the identification thereof, any reference hereinbefore and hereinafter to the free compounds shall be understood as including the corresponding salts, where appropriate and expedient.

Where compounds or salts are mentioned, this is meant to include also the singular (one compound or salt).

The compounds of formula I have valuable pharmacological properties and can be used, for example, as drugs to treat proliferative diseases.

The compounds of formula I inhibit the proteasome activity. It is known that proteins targeted for the degradation by the multicatalytic proteasome complex have inter alia functions in the cell-cycle control (e.g. cyclins, p21, p27) and apoptosis (e.g. p53) (Rolfe, M., Chiu, I. M. and Pagano, M., The ubiquitin-mediated proteolytic pathway as therapeutic area. J. Mol. Med. 75, 1997, 5-17). Inhibitors of the proteasome are therefore suitable for the treatment of proliferative diseases which respond to the inhibition of the proteasome activity. Proliferative diseases like psoriaris and tumors, in particular solid tumors like colon tumor, breast tumor, lung tumor and prostate tumor belong to the diseases to be mentioned here. Other proliferative diseases to be treated are psoriasis or restenosis. Also further diseases can be treated in a broader aspect of the invention, e.g. muscle protein degradation or other diseases connected with intracellular protein degradation or negaive nitrogen balance, e.g. in patients suffering from sepsis, bums, trauma, cancer, chronic or systemic infections, neuromotor degenerative disease, e.g. muscular dystrophia, acidosis, spinal or nerve injuries, during corticosteroid treatment, or the like;, diseases related to antigen presentation on cells; diseases related to cell adhesion; or the like, especially as far as proteasome inhibition is effective.

Inhibition of 20S Proteasome

The compounds of formula I inhibit the 20S proteasome, especially with high selectivity the chymotrypsin-like activity.

The multicatalytic proteasome complex is responsible for the ATP-dependent proteolysis of most cellular proteins. Although the 20S proteasome contains the proteolytic core, it cannot degrade proteins in vivo unless it is complexed with 19S caps, at either end of its structure, which itself contains multiple ATPase activities. This larger structure is known as the 26S proteasome and will rapidly degrade proteins that have been targeted for the degradation by the addition of multiple molecules of the 8.5 kDa polypeptide ubiquitin. As mentioned above, proteins targeted for proteasomal degradation have functions in the cell-cycle. The compounds of formula I are therefore highly suitable for the treatment of diseases which respond to inhibition of the activity of the 20S proteasome, which is the case for the proliferative diseases (or further the other diseases) mentioned above.

The inhibition of the chymotrypsin-like activity of the 20S proteasome can be demonstrated by the following experiment. It is based on the hydrolysis of the fluorogenic peptide Suc-LLVY-AMC (Succinyl-leucine-leucine-valine-tyrosine-7-amino-4-methyl-coumarin), which is cleaved exclusively at the Y-AMC bond by the 20S proteasome. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (λ_(ex) (excitation wavelength): 355 nm; λ_(em) (emission wavelength): 460 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis product Suc-LLVY.

2 μl of a 1 mM solution of a test compound in DMSO (dimethylsulfoxide, 20 μM final concentration in the well) are preincubated for 60 min at room temperature in black 96-well microtiter plates together with a mixture of 1 μl purified human placenta 20S proteasome (Diabetes Forschungsinstitut, Düsseldorf, Germany; about 100 ng proteasome, depending on the proteasome preparation) and 47 μl buffer-Ca (5 mM CaCl₂, 20 mM Tris/HCl (Tris-(hydroxymethyl)-amino-methane hydrochloride) pH 8.0). 3 μl of a 2.67 mM solution of Suc-LLVY-AMC (Bachem, Switzerland ) in DMSO (80 μM final concentration in the well) and 47 μl buffer-Ca are mixed and added. The resulting mixture is incubated for 3-24 h at 37° C. If desired, proteolysis of the substrate can be stopped by addition of 50 μl stop solution (100 mM monochloroacetic acid, 130 mM NaOH, 100 mM acetic acid, pH=4.3). The fluorescence is monitored with a FLUOROSKAN ASCENT™ microtiter plate reader.

For each measurement series two control experiments are carried out:

-   -   1.) 0% value: 2 μl DMSO are used in the above described assay         instead of 2 μl of a 1 mM solution of a test compound in DMSO         and 48 μl A buffer-Ca instead of a mixture of 47 μl buffer-Ca         and 1 μl purified proteasome.     -   2.) 100% value: in the above described assay 2 μl DMSO are used         instead of 2 μl of a 1 mM solution of the test compound in DMSO.

Calculation $\begin{matrix} {\%\quad{remaining}} \\ {activity} \end{matrix} = {{\frac{\left( {{{value}\quad{with}\quad{compound}} - {0\%\quad{value}}} \right)}{\left( {{100\%\quad{value}} - {0\%\quad{value}}} \right)} \cdot 100}\%}$

The IC₅₀ value is defined as that concentration of a compound at which the remaining activity is 50% compared to the 0% control. Compounds of formula I exhibit an IC₅₀ value for the inhibition of the chymotrypsin-like activity of the 20S proteasome in the range of between 0.3 nM and 1 μM, especially between 0.5 nM and 200 nM.

On the other hand, the compounds according to the invention are highly specific for the chymotrypsin-like activity.

The inhibition of the trypsin-like (TrpL) activity and the peptidoglutamyl-hydrolyzing (PGPH) activity of the 20S proteasome can be demonstrated by monitoring the hydrolysis of the fluorogenic peptides Boc-LRR-AMC (Boc-leucine-arginine-arginine-7-amino-4-methyl-coumarin) for the TrpL activity and the hydrolysis of Z-LLE-AMC (Z-leucine-leucine-glutamate-7-amino-4-methyl-coumarin) for the PGPH activity. The peptides are cleaved exclusively at the amino acid-AMC bond by the corresponding proteolytic activity of the 20S proteasome. Hydrolysis of the peptides is accompanied by an increase in fluorescence intensity (λ_(ex)=355 nm, λ_(em)=460 nm) caused by the release of the internally quenched 2-aminobenzoyl fluorescence, as already described.

2 μl of a 1 mM solution of a test compound in DMSO (dimethylsulfoxide, 20 μM final concentration in the well) are preincubated for 60 min at room temperature in black 96-well microtiter plates together with a mixture of 1 μl human 20S proteasome (about 100 ng proteasome purified from human erythrocytes as described by Dahlmann et al., Biochem. J. 309, 195-202 (1995) and McGuire et al., Biochim. Biophys. Acta 995(2), 181-186 (1989)) and 47 μl buffer-Ca (5 mM CaCl₂, 20 mM Tris/HCl (tris(hydroxymethyl)-amino-methane hydrochloride) pH 8.0). 3 μl of a 2.67 mM solution of Suc-LLVY-AMC (Bachem, Switzerland) in DMSO (80 μM final concentration in the well) and 47 μl buffer-Ca are mixed and added for assaying ChyL activity. 3 μl of a 1.67 mM solution of Boc-LRR-AMC (Affinity, Mamhead, UK) in DMSO (50 μM final concentration in the well) or 3 μl of a 3.67 mM solution of Z-ILE-AMC (Calbiochem/Juro Supply AG, Lucerne, Switzerland) in DMSO (80 μM final concentration in the well) and 47 μl buffer-Ca are mixed and added for assaying TryL or PGPH activity, respectively. The resulting mixture is incubated for 3 to 24 h at 37° C. If desired, proteolysis of the substrate can be stopped by addition of 50 μl stop solution (100 mM monochloroacetic acid, 130 mM NaOH, 100 mM acetic acid, pH=4.3). The fluorescence is monitored with a FLUOROSKAN ASCENT™ microtiter plate reader.

To each measure series two control experiments have to be carried out:

-   -   1) 0% value: 2 μl DMSO are used in the above described assay         instead of 2 μl of a 1 mM solution of a test compound in DMSO         and 48 μl buffer-Ca instead of a mixture of 47 μl buffer-Ca and         1 μl purified proteasome.     -   2) 100% value: In the above described assay 2 μl DMSO are used         instead of 2 μl of a 1 mM solution of the test compound in DMSO.         ${{Calculation}:\begin{matrix}         {\%\quad{remaining}} \\         {activity}         \end{matrix}} = {{\frac{\left( {{{value}\quad{with}\quad{compound}} - {0\%\quad{value}}} \right)}{\left( {{100\%\quad{value}} - {0\%\quad{value}}} \right)} \cdot 100}\%}$

The IC₅₀ value is defined as that concentration of a compound at which the remaining activity is50%.

In these test systems, the selectivity of the compounds of the formula I for the chymotrypsin-like activity is more than 20-fold, preferably 50 to 3000-fold higher than that for the trypsin-like or peptidoglutamyl-hydrolyzing activity, this selectivity being a further advantage of the compounds of formula I.

The antiproliferative activity of the compounds of formula I can be demonstrated in vitro against e.g. the human breast carcinoma cell line MDA-MB435 (HTB-129) obtained from the American Type Culture Collection (ATCC, Rockville, USA). Routinely the ‘CellTiter™’ proliferation assay (Promega, Madison Mich.) is used following the procedure recommended by the supplier. This assay is performed in 96well plates prepared for tissue culture. Cells are seeded in a density of 2.5×10⁴ per well in 50 μl complete MEM [minimal essential medium] (Gibco-LifeTechnologies) supplemented with 10% fetal calf serum, 100 U/mI PenStrep, 1 mM sodium pyruvate, 4 mM L-Glutamine, 20 mM HEPES and Non Essential Amino Acids. Cells are incubated for 24 hours at 37° C. in humidified atmosphere equilibrated with 5% CO₂. Test compounds are added to the cell supernatant as serial dilutions in 50 μl MEM-complete per well. Cells are incubated for at least 48 hours at 37° C. in humidified atmosphere equilibrated with 5% CO₂. The tetrazolium dye MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is added to the cell supernatant in a volume of 15 μl per well and cell cultures are incubated for 4 hours. Thereafter, the reaction is stopped by addtion of 100 μl stopsolution per well and the plates are incubated for one additional hour. The conversion of the tetrazolium dye by metabolically active cells yields soluble formazan. The absorbance of the blue colored cell supernatant is proportional to the amount of viable cells. The absorbance is monitored at the wavelenght of 550 and 630 nm using a microtiter plate reader (Dynatech MR5000).

Serial dilutions of compound are prepared by first diluting the 10 mM compound stock solution (in DMSO) to a 60 μM test solution in MEM-complete followed by nine successive 1:3 dilutions in MEM-complete. Wells containing MEM-complete serve as negative control, background (0%). Wells containing cells and MEM/0.6% DMSO serve as positive control (100%). The % remaining activity is determined by calculating as described above for the inhibition of the chymotrypsin-like activity of the 20S proteasome. The IC₅₀ value is defined as that concentration of a compound at which the remaining activity is 50%.

Compounds of formula I exhibit an IC₅₀ value for the antiproliferative activity in the range of between 0,5 nM and 1 μM, especially between 1 nM and 200 nM.

The antitumoral action of the compounds of formula I can also be demonstrated in vivo:

In vivo Evaluation of Antitumor Action in Nude Mice Using Human Tumor Xenografts

Female or male BALB/c nu/nu (Novartis animal farm, Sisseln, Switzerland or Bomholtgaard, Copenhagen, Denmark) mice with subcutaneously transplanted human tumors are used for the evaluation of the antitumor action of the compounds of formula I against cell lines originating from the four tumor types, breast tumor: MCF-7; lung tumor NCl H596: colon tumor: HCT 116; and prostate tumor PC 3.

Materials:

Human colon carcinoma HCT 116 (ATCC CCL 247), human squamous cell lung carcinoma NCl H596 (ATCC HTB 178), estrogen-dependent breast carcinoma MCF-7 (ATCC HTB 22), and the human prostate cancer PC 3 cell line are obtained from the American Type Culture Collection (ATCC, Rockville, USA). The cells are cultured at 37° C. in a 5% vV CO₂ and 80% relative humidity atmosphere in the following media: NCl H 596: RPMI 1640, 20% v/v FBS (fetal bovine serum), 1% w/v glutamine; HCT-116: McCoy's 5A, 10% v/v FBS, 1% w/v glutamine; PC 3: RPMI 1640, 20% v/v FBS, 1% w/v glutamine; MCF-7, RPMI 1640, 20% v/v FBS, 1% w/v glutamine. All of these cell lines are adherent and can be released by rinsing with Hank's balanced salt and treatment with 0.25% w/v trypsin. All of these cells are prepared as master cell stocks (in culture media supplemented to contain 20% v/v FBS and 7% v/v DMSO) and stored at −125° C. (liquid nitrogen vapors). Working cell stocks are prepared from the master stocks by thawing and expanding the cells over three passages, which are then distributed to vials and frozen. Viability (trypan blue exclusion test using 0.5% w/v trypan blue) prior to freezing is >90% for all cell lines.

Establishing Solid Tumors in Nude Mice:

Mice are kept under controlled conditions (Optimal Hygienic Conditions, [OHC]) with free access to sterile food and water. Tumors are established after subcutaneous injection of cells (a minimum of 2×10⁶ cells in 100 μl PBS (phosphate buffered saline) or medium) in carrier mice (4-8 mice per cell line). Injections are made s.c. in the left flank of the mouse mid-way between the tail and head. The resulting tumors are serially passaged for a minimum of three consecutive transplantations prior to start of treatment. Tumors are transplanted when the tumor reaches a volume of 800 to 1000 mm³.

Transplanting Solid Tumors in Nude Mice:

Donor mice are anesthetized (Forene®, Abbott, Switzerland) and killed by cervical dislocation. The skin is disinfected and the tumor removed by dissection. The outer edges of the tumor mass is removed using a scalpel, and the resulting mass is trimmed into pieces of about 3-4 mm in height. Sections of 3-4 mm² are cut and placed into sterile 0.9% w/v NaCl. Sections of tumor that are necrotic are not used.

Tumor fragments are implanted s.c. into the left flank of the recipient mice. Recipient mice are anesthetized (Forene®, Abbott, Switzerland) and the skin on the entire back and sides of the mice is disinfected. The skin 0.5 to 1 cm above the tail is raised and a single 1 to 1.5 cm incision is made. Tumor sections are pushed into a 13-gauge trocar needle. The trocar needle is pushed into the opening of the skin, and advanced under the skin to a point midway between the head and the tail. The tumor fragment is deposited by advancing the trocar. The wound is sutured using one or two metal clips.

In the case of estrogen-dependent breast tumors, estrogen pellets (17b-estradiol, 5 mg/pellet giving a sustained release over 60 days are obtained from Innovative Research of America, Sarasota, USA), are placed subcutaneously in the other flank.

The tumors are allowed to increase until the size is 100 to 150 mm³ before treatment is started. The tumors are then measured and placed into groups (normally n=6 to 8) that are balanced according to the mean size and range of tumor volumes. Groups are randomly assigned to treatment groups.

Preparation and Application of Test Compounds:

Stock solutions of 40 mg/ml of compound are dissolved in 100% DMSO and stirred at room temperature until a clear solutions is obtained. Prior to each administration, 10% Tween® 80 (Fluka, Buchs, Switzerland; polyoxyethylene-sorbitane-monostearate; Tween® is a trademark from ICI Americas Inc., USA) is added to the stock solution and then diluted 1:20 (v/v) with sterile 0.9% w/v NaCl or water. Solutions and dilutions are prepared daily prior to application. Applications are given 7 days a week (p.o., i.p., s.c. or i.v.). The volumes of application are: p.o., 25 ml/kg; i.p., 25 ml/kg; s.c., 10 ml/kg; i.v., 10 ml/kg.

Measurement of tumor volumes: Tumor growth is monitored once, twice or three times weekly (depending on the growth rate of the tumor line) and 24 hours after the last treatment by measuring perpendicular diameters. Calipers capable of determining mm distances are used. Tumor volumes are calculated according to the formula L×D²×π/6 (L: length; D: diameter). Antitumor activity is expressed as T/C % (mean increase of tumor volumes of treated animals divided by the mean increase of tumor volumes of control animals multiplied with 100). Tumor regression (%) represents the smallest mean tumor volume compared to the mean tumor volume at the start of treatment. Delta (Δ) tumor volumes compared the change in tumor volume during the duration of the experiment (volume on the last treatment day minus volume on the first treatment day). Any animals in which the tumor reaches a size exceeding approximately 1500 to 2000 mm³ are sacrificed.

Additional Measurements:

Body weights and survival data are also collected. Delta (Δ) body weights are calculated as an indication of tolerability to treatment (weight on the last treatment day—weight on the first treatment day). Statistically significant body weight loss, or mortalities, are considered indicators of poor tolerability to treatment. Additionally, mice are monitored once or twice daily for general health.

Statistical Analyses:

The basic approach for statistical analyses is to use tests for multiple comparisons to judge the statistical significance of differences between treatment groups, and differences within a group to determine if treatment induced stable disease or tumor regressions. As subcutaneous tumor volumes are not always normally distributed, differences in the subcutaneous tumor volumes between treatment groups is determined using the non-parametric Kruskal-Wallis one way ANOVA test on ranked data and the statistical significance of differences between treatment groups as compared to control groups determined using the Dunnett test. Pair wise comparisons between all groups is performed using the Student-Newman-Keuls (SNK) method. If only two groups are compared, the Rank sum test is used. Animal body weights are normally distributed, and changes in body weights within a group are analyzed by paired t-tests and between group differences are analyzed by a One-Way ANOVA and pair-wise comparisons made using the Tukey test. For all tests the level of significance is set at p<0.05.

Using the same methods, instead of the mentioned tumor cell lines also the estrogen-independent breast tumor cell line MDA-MB435 (HTB-129), obtainable form the American Type Culture Collection, can be used. Culture of this cell line is in MEM, 10% v/v FBS, 1% w/v glutamine, adherence and release as described above for the other cell lines.

Another in vivo test to determine the antitumoral action of the compounds of formula I is the following hollow fiber assay:

Hollow Fiber Assay: Evaluation of Antitumor Action in Nude Mice

In this assay four different human tumors encapsulated in hollow fibers are implantated subcutaneously and/or intraperitonealy into nude mice (athymic female outbred nude mice (Ncr nu/nu)). Animals are then treated with a test compound formulated in an appropriate vehicle, while control animals are treated with the vehicle alone. At the end of the experiment fibers are retrieved, and the number of viable cells is measured using a metabolic assay (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Activity of the test compound is measured by comparing the growth of cells in the experimental animals (T) to the growth of cells in the control animals treated with the vehicle alone (C), and is expressed as %T/C.

Materials:

Human colon carcinomas SW 620 and LS 174T, and breast carcinoma MDA-MB435S are obtained from the American Type Culture Collection (ATCC, Rockville, USA). The colon carcinoma MIP 101, expressing high levels of pgp-1, was originally established from a patient at the Dana Farber Cancer Institute, Boston, USA. All cell lines are grown according to standard tissue culture techniques in RPMI 1640 containing the following additives: 5% FBS (Fetal Bovine Serum), 5 mg/ml insulin, 5 mg/ml transferrin, 5 mg/ml selenous acid, 1 nM β-estradiol, 1 nM testosterone.

Design of the Assay:

The human solid tumor cell lines are encapsulated in PVDF (polyvinylidene fluoride) hollow fibers that are permeable to molecules<500,000 Daltons and that have an inner diameter of 1 mm (Biopore, Spectrum Medical, Calif., USA). After encapsulation and prior to implantation into animals, cells are allowed to attach to the inner surface of the fiber by incubation in the tissue culture media for 24 hours. Four hollow fibers are then implanted into one animal intraperitoneally or subcutaneously. In the experimental setup four to six animals are used per group, and the experiment consists of a minimum of three groups:

-   -   1. “Time 0” group     -   2. Control (placebo) group     -   3. “Treated” (tested compound) group.

Treatment starts 24 hours after implantation of the fibers. Animals are treated once daily, at days 1, 3, and 5. At the same time when the treatment starts, the animals from “Time 0” group are sacrificed, fibers are retrieved, and the number of viable cells at the beginning of the experiment is determined (T₀). At day 7 after the implantation all animals are sacrificed, fibers are retrieved, and the number of viable cells is determined for the Control (C_(v)), and for the “Treated” (T_(v)) groups. %T/C is then calculated according to the formula: %T/C=(T _(v) −T _(o))/(C _(v) −T ₀)×100%

Encapsulation of Tumor Cells in Hollow Fibers:

Fibers are cut into desired length and soaked for at least 72 h in 70% Ethanol. Afterwards, fibers and instruments for the encapsulation are sterilized. The human solid tumor cell line grown in tissue culture is trypsinized, suspended in a small amount of tissue culture media and transferred into the fibers using a syringe. The fibers are heat sealed and incubated at 37° C. for 24 h in an atmosphere comprising 5% CO₂. The fibers are then implantated subcutaneously and/or intraperitonealy into nude mice.

Determination of Viable Cells in Hollow Fibers:

1 g MTT is added to 200 ml PBS (phosphate-buffered saline), stirred for 20 min and filtered (0.22 micron filter). 10 ml of this solution are mixed with 40 ml RPMI 1640 with additives to give the MTT working solution. The retrieved fibers are incubated in 5 ml RPMI 1640 for 30 min at 37° C. in 5% CO₂ for stabilization. 0.5 ml of MTT working solution are added to each well of the sample plate. The plates are incubated at 37° C. in 5% CO₂ for 4 h. The MTT is aspirated out of each well in the sample plate. 2 ml of 2.5% aqueous protamine sulfate solution is added to each well of the sample plate. The plates are incubated at 4° C. for 24 h. The protamine sulfate is aspirated out of each well in the sample plate. 2 ml of protamine sulfate are added to each well and the plates are incubated at 4° C. for further 2-4 h. Each fiber is transfered to a well in a 24 well plate. The fibers are cut short allowing them to lie on the bottom of the wells and dried overnight. 250 μl of DMSO are added to each well. The plates are placed on an orbital shaker for 4 h with a cover to protect the MTT from light. 150 μl of each sample are transfered to the appropriate well in a 96 well plate. The plates are read at 540 nm using DMSO as the blanking well.

The bioavailability after oral administration of compounds of formula I can be shown e.g. in the following test: For peroral administration, a solution of the test substance (25 mg/ml) in a suitable solvent such as Cremophor RH40®/Maisine®/propylene glycol/ethanol (38/32/15/15) is prepared. Female Balb/c mice are fasted for 24 hours prior to the start, and throughout the experiment water is given ad libitum. At various times following drug administration, blood samples are obtained by sacrifycing animals under anaesthesia by cutting the vena jugularis, followed by cervical dislocation. Samples are collected in heparinized tubes (typically 0.4-0.6 ml). For sample analysis solid phase extraction and HPLC are used. Drug concentration in the samples is calculated by least-squares linear regression analysis of the peak area ratio (inhibitor/internal standard) of spiked blood standards versus concentration. From the concentration versus time data, the “Area Under the Curve” (AUC) value is calculated by the trapezoidal rule.

PREFERRED EMBODIMENTS OF THE INVENTION

In the following preferred embodiments of the invention, general definitions or expressions can, individually, independently by several or all completely, be replaced with more specific definitions as given above, if not mentioned otherwise, thus defining even more preferred embodiments of the invention.

Preference is given to compounds of formula I, wherein

-   -   R₁ is either substituted aryl-lower alkylcarbonyl or         unsubstituted or substituted aryl,     -   R₂ is substituted aryl or unsubstituted or substituted         heterocyclyl,     -   R₃ is lower alkyl, unsubstituted or substituted aryl or lower         alkyl which is substituted by unsubstituted or substituted aryl,     -   R₄ is a moiety of the formula IA given above wherein A₁ and A₂         are hydroxy, lower alkyloxy, aryloxy with unsubstituted or         substituted aryl or cycloalkyloxy with unsubstituted or         substituted cycloalkyl, or wherein A₁ and A₂, together with the         binding boron atom and the two binding oxygen atoms form a ring         of the formula IA* given above wherein W is unsubstituted or         substituted lower alkylene bound via two different carbon atoms         that are spatially nearby or vicinal, especially in vicinal or,         relatively to each other, in “1,3”-position, and     -   R₅ is lower alkyl,     -   or salts thereof.

More preference is given to compounds of the formula I wherein

-   -   R₁ is phenyloxyphenyl-lower alkylcarbonyl; phenyl-lower         alkoxyphenyl-lower alkylcarbonyl; pyridyloxyphenyl-lower         alkylcarbonyl; phenyl-lower alkylcarbonyl substituted by lower         alkoxy, especially methoxy, halogen, especially fluoro or         chloro, or halogen-lower alkyl, especially trifluoromethyl; or         preferably unsubstituted or substituted phenyl or naphthyl,         wherein in both cases the substituents if present are         independently one or more, especially one to three, substituents         selected from the group consisting of lower alkyl, hydroxy,         lower alkoxy, lower alkanoyloxy, carboxy, lower alkoxycarbonyl,         formyl, lower alkanoyl, amino, N-lower alkylamino, N,N-di-lower         alkylamino, mercapto, sulfo, lower alkyl-thio, carbamoyl, slower         alkyl-carbamoyl; N,N-di-lower alkyl-carbamoyl, phenyl, naphthyl,         pyridyl, cyano and nitro, more preferably lower alkoxy alkoxy,         especially methoxy or ethoxy;     -   R₂ is phenyl substituted by one or more, especially one to         three, moieties independently selected from the group consisting         of hydroxy, lower alkoxy, especially methoxy, halogen,         especially fluoro or chloro, and halogen-lower alkyl, especially         trifluoromethyl;     -   R₃ is lower alkyl, especially isobutyl, phenyl or phenyl         substituted by one or more, especially up to three substituents         independently selected from the group consisting of hydroxy,         lower alkoxy, especially methoxy, halogen, especially fluoro or         chloro, and halogen-lower alkyl, especially trifluoromethyl;     -   R₄ is —B(OH)₂ (especially preferred) or or         2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]-dec-4-yl,         especially (1S, 2S, 6R,         8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl;         and     -   R₅ is lower alkyl, especially isopropyl;     -   or salts thereof.

Preference is given especially to compounds of formula I, wherein

-   -   R₁ is phenyloxyphenylacetyl, benzyloxyphenylacetyl,         pyridyloxyphenylacetyl, biphenylyl, pyridylphenyl, lower         alkylphenyl or substituted phenylpropionyloxy wherein the phenyl         substituents are up to three substituents independently selected         from the group consisting of methoxy, fluoro, chloro and         trifluoromethyl;     -   R₂ is phenyl substituted with up to three methoxy substituents,         especially 2,3,4-trimethoxyphenyl or 3,4,5-trimethoxyphenyl;     -   R₃ is isobutyl or phenyl that is unsubstituted or substituted         with up to three moieties independently selected from hydroxy,         fluoro and methoxy;     -   R₄ is (1S, 2S, 6R,         8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl         or especially —B(OH)₂; and     -   R₅ is isopropyl;     -   or salts thereof.

Preference is further given especially to compounds of formula I, wherein

-   -   R₁ is biphenylyl, lower alkyl-phenyl, phenyl-lower         alkyl-carbonyl, phenoxy-phenyl-lower alkyl-carbonyl,         phenyl-lower alkoxy-phenyl-lower alkyl-carbonyl or         pyridyl-phenyl;     -   R₂ is either phenyl substituted by 1 to 3 lower alkoxy radicals         or phenyl-lower alkoxy-phenyl;     -   R₃ is lower alkyl or phenyl-lower alkyl;     -   R₄ is 4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl,         (1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl         or —B(OH)₂; and     -   R₅ is lower alkyl;     -   or salts thereof.

Very special preference is given to compounds of formula I or salts thereof, wherein the stereochemistry is as depicted in formula I*

wherein the shown configuration represents the absolute configuration and wherein R₁, R₂, R₃, R₄ and R₅ have the meanings as defined for a compound of formula I, especially those meanings described hereinabove as being preferred.

Also preferred are compounds of formula I or salts thereof, wherein the stereochemistry is as depicted in formula I**

wherein the shown configuration represents the absolute configuration and wherein R₁, R₂, R₃, R₄ and R₅ have the meanings as defined for a compound of formula I, especially those meanings described hereinabove as being preferred.

Further preferred are also compounds of formula I or salts thereof, including mixtures of diastereomers, wherein the stereochemistry is as depicted in formula I***

wherein the shown configuration represents the absolute configuration and wherein R₁, R₂, R₃, R₄ and R₅ have the meanings as defined for a compound of formula I, especially those meanings described hereinabove as being preferred.

Most especially preferred are the compounds of formula I described in the Examples, or pharmaceutically acceptable salts thereof.

The compounds of formula I or salts thereof are prepared in accordance with processes known per se, though not previously described for the manufacture of the compounds of the formula I. The processes preferably comprise

-   -   a) reacting a dipeptide analogue of the formula II,         wherein R₃, R₄ and R₅ have the meanings given under formula I,         with an amino acid of the formula III,         or a reactive derivative thereof, wherein R₁ and R₂ have the         meanings given under formula I,     -   functional groups present in a compound of formula II and/or         III, with the exception of the groups participating in the         reaction, being protected if necessary by readily removable         protecting groups, and any protecting groups present are         removed,         or     -   b) for the production of a compound of the formula I wherein R₁         is arylalkylcarbonyl or heterocyclylalkylcarbonyl and the other         moieties R₂ to R₅ have the meanings given under formula I,         reacting an amino compound of the formula IV,         wherein R₂; R₃, R₄ and R₅ have the meanings given under formula         I, with a carbonic acid of the formula V,         or a reactive derivative thereof, wherein R₁ is         arylalkylcarbonyl or heterocyclylalkylcarbonyl, functional         groups present in a compound of formula IV and/or V, with the         exception of the groups participating in the reaction, being         protected if necessary by readily removable protecting groups,         and any protecting groups present are removed,     -   and, if desired, converting a compound of formula I obtained by         process a) or b) into another compound of formula I, converting         an obtained free compound of formula I into a salt, converting         an obtained salt of a compound of formula I into a different         salt or into its free form, and/or separating a mixture of         isomeric compounds of formula I into the individual isomers.

The different possible stereoisomers of compounds of formula I can be prepared by using educts with the appropriate configuration. For example, compounds of formula I* or salts thereof can be prepared by

-   -   a) reacting a dipeptide analogue of the formula II*,         wherein R₃, R₄ and R₅ have the meanings given under formula I,         with an amino acid of the formula III*,         or a reactive derivative thereof, wherein R₁ and R₂ have the         meanings given under formula I, functional groups present in a         compound of formula II* and/or III*, with the exception of the         groups participating in the reaction, being protected if         necessary by readily removable protecting groups, and any         protecting groups present are removed,         or     -   b) for the production of a compound of the formula I* wherein R₁         is arylalkylcarbonyl or heterocyclylalkylcarbonyl and the other         moieties R₂ to R₅ have the meanings given under formula I,         reacting an amino compound of the formula IV*,         wherein R₂, R₃, R₄ and R₅ have the meanings given under formula         I, with a carbonic acid of the formula V,         or a reactive derivative thereof, wherein R₁ is         arylalkylcarbonyl or heterocyclylalkylcarbonyl, functional         groups present in a compound of formula IV* and/or V, with the         exception of the groups participating in the reaction, being         protected if necessary by readily removable protecting groups,         and any protecting groups present are removed,     -   and, if desired, converting a compound of formula I* obtained by         process a) or b) into another compound of formula I*, converting         an obtained free compound of formula I* into a salt, or         converting an obtained salt of a compound of formula I* into a         different salt or into its free form.

General Remarks:

The end products of formula I may contain substituents that can also be used as protecting groups in starting materials for the preparation of other end products of formula I, e.g. in the case of R₄ other than —B(OH)₂. Thus, within the scope of this text, only a readily removable group that is not a constituent of the particular desired end product of formula I is designated a “protecting group”, unless the context indicates otherwise.

The protection of functional groups by such protecting groups, the protecting groups themselves, and their cleavage reactions are described for example in standard reference works, such as J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der organischen Chemie” (Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15/l, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosäuren, Peptide, Proteine” (Amino acids, Peptides, Proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide und Derivate” (Chemistry of Carbohydrates: Monosaccharides and Derivatives), Georg Thieme Verlag, Stuttgart 1974.

A characteristic of protecting groups is that they can be removed readily (i.e. without the occurrence of undesired secondary reactions) for example by solvolysis, reduction, photolysis or alternatively under physiological conditions (e.g. by enzymatic cleavage).

Removal of a protecting group for the —B(OH)₂-group (in order to obtain a compound of the formula I wherein R₄ is —B(OH)₂) preferably takes place with an acid, e.g. hydrogen chloride, in an appropriate solvent, e.g. a lower alkanol, such as methanol, or a lower alkane, such as hexane, or a mixture thereof, at temperatures of 0 to 50° C., e.g. at room temperature.

Process a):

The reaction is carried out by dissolving the compounds of formulae II and III in a suitable solvent, for example N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, methylene chloride, or a mixture of two or more such solvents, and by the addition of a suitable base, for example triethylamine, diisopropylethylamine (DIEA) or N-methylmorpholine and a suitable coupling agent that forms a preferred reactive derivative of the carbonic acid of formula III in situ, for example dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBT); O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TPTU); O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU); or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). For review of other possible coupling agents, see e.g. Klauser; Bodansky, Synthesis 1972, 453-463. The reaction mixture is preferably stirred at a temperature of between approximately −20 and 50° C., especially between 0° C. and room temperature, to yield a compound of formula I. The reaction is preferably carried out under an inert gas, e.g. nitrogen or argon.

Process b):

The reaction is preferably carried out under conditions analogous to those described for process a).

Salts of a compound of formula I with a salt-forming group may be prepared in a manner known per se. Acid addition salts of compounds of formula I may thus be obtained by treatment with an acid or with a suitable anion exchange reagent.

Salts can usually be converted to free compounds, e.g. by treating with suitable basic agents, for example with alkali metal carbonates, hydrogencarbonates, or hydroxides, typically potassium carbonate or sodium hydroxide.

Stereoisomeric mixtures, e.g. mixtures of diastereomers, can be separated into their corresponding isomers in a manner known per se by means of suitable separation methods. Diastereomeric mixtures for example may be separated into their individual diastereomers by means of fractionated crystallization, chromatography, solvent distribution, and similar procedures. This separation may take place either at the level of one of the starting compounds or in a compound of formula I itself. Enantiomers may be separated through the formation of diastereomeric salts, for example by salt formation with an enantiomer-pure chiral acid, or by means of chromatography, for example by HPLC, using chromatographic substrates with chiral ligands.

Compounds of the formula I wherein R₄ is other than —B(OH)₂ can be converted into compounds of the formula I wherein R₄ is —B(OH)₂ according to standard procedures, e.g. using isobutyl-boronic acid (i-BuB(OH)₂ in the presence of an acid, especially hydrohalic acid in a water/methanol/hexane mixture, at temperatures preferably ranging from 0 to 50° C., e.g. at room temperature.

In both process a) and b), for the conversion or for the synthesis of the intermediates or starting material, especially as described below, the solvents from which those can be selected which are suitable for the reaction in question include for example water, esters, typically lower alkyl-lower alkanoate, e.g diethyl acetate, ethers, typically aliphatic ethers, e.g. diethyl-ether, or cyclic ethers, e.g. tetrahydrofuran, liquid aromatic hydrocarbons, typically benzene or toluene, alcohols, typically methanol, ethanol or 1- or 2-propanol, nitriles, typically acetonitrile, halogenated hydrocarbons, typically dichloromethane, acid amides, typically dimethylformamide, bases, typically heterocyclic nitrogen bases, e.g. pyridine, carboxylic acids, typically lower alkanecarboxylic acids, e.g. acetic acid, carboxylic acid anhydrides, typically lower alkane acid anhydrides, e.g. acetic anhydride, cyclic, linear, or branched hydrocarbons, typically cyclohexane, hexane, or isopentane, or mixtures of these solvents, e.g. aqueous solutions, unless otherwise stated in the description of the process. Such solvent mixtures may also be used in processing, for example through chromatography or distribution. New starting materials and/or intermediates, as well as processes for the preparation thereof, are likewise the subject of this invention. In the preferred embodiment, such starting materials are used and reaction conditions selected such as to allow the manufacture of the preferred compounds.

The starting materials of formulae II-V or their precursors are known, can be prepared according to known processes, or are commercially obtainable; in particular, they can be prepared using processes identical or in analogy to those described in the Examples.

A compound of formula II, wherein the substituents are as defined above under formula I, is obtainable for example by the following reactions:

First, a boronic acid analogue of an amino acid of the formula VI

comprising for example the configuration as indicated in formula VI*

wherein R₃ has the meanings given above for compounds of formula I and R₄ has the meanings other than —B(OH)₂ mentioned above for compounds of formula I, especially is (1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl, or an acid addition salt thereof, especially the salt thereof with trifluoroacetic acid, is condensed with an amino acid of the formula VII

comprising for example the configuration as indicated in formula VII*

or a reactive derivative thereof, wherein R₅ has the meanings given above for compounds of the formula I and Pr₁ is a protected amino group, preferably tert-butoxycarbonylamino, under reaction conditions analogous to those described for reaction a) above (also a condensation reaction, also preferably with in situ formation of active carbonic acid derivatives), thus yielding a compound of formula II in N-protected form which is then N-deprotected, e.g. using conditions described in the standard textbooks mentioned above, in the case of tert-butoxycarbonylamino e.g. with hydrochloric acid in an appropriate solvent, e.g. dioxane and/or methylene chloride giving a compound of the formula II that can be used directly in process a).

The boronic acids of the formula VI are known, commercially available and/or can be synthesized according to known procedures. For example, compounds of the formula VI wherein R₃ is lower alkyl, especially isobutyl and R₄ is as described for compounds of the formula VI, preferably (1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl, can be prepared by reacting a compound of the formula VIII,

wherein R₄ has the meanings just described, in an appropriate solvent, e.g. methylene chloride, with n-lower alkyl lithium, especially n-butyllithium, and subsequently with zinc chloride, yielding a compound of the formula IX,

wherein R₄ has the meanings given above under formula VI. This compound is then reacted with LiN(SiCH₃)₂, and the resulting compound of the formula is then reacted in the presence of trifluoro acetic acid to yield the salt of the formula X,

wherein R₄ has the meanings given under formula VI, which is a compound of the formula VI and can then be used directly for reaction with the compound of formula VII as shown above.

A compound of the formula III is known, commercially available and/or can be obtained according to standard procedures.

For example, a compound of the formula III wherein R₁ is aryl, especially biphenylyl, may be prepared by reacting a compound of the formula XI,

comprising for example the configuration as indicated in formula XI*

wherein R₂ has the meanings given for a compound of the formula I, which is known, commercially available or obtainable according to standard procedures, with a compound of the formula XII, R₁—X   (XII) wherein R₁ is aryl and X is halogen, especially bromo, in an appropriate solvent; e.g. in dimethylformamide, in the presence of a base, especially an alkali metal carbonate, e.g. potassium carbonate, at temperatures between 50 and 100° C., e.g. at 90° C., preferably under inert gas, e.g. nitrogen or argon. This directly yields the corresponding compound of the formula III.

Amino acid derivatives of the formula VII are known, commercially available or obtainable according to standard procedures. They are preferably used in the amino protected form, e.g. with tert-butoxycarbonylamino instead of the free amino group.

Compounds of the formula IV can be obtained e.g. by reacting a compound of the formula II comprising for example the configuration as indicated in formula II*, as defined in process a), with an N-protected amino acid of the formula XIII,

comprising for example the configuration as indicated in formula XIII*

or a reactive derivative thereof, wherein R₂ is as defined under formula I and Pr₂ is protected amino, especially tert-butoxycarbonylamino, under preferred condensation reaction conditions as described under process a) above. From the resulting compound, a compound of formula IV wherein the N-terminal amino group is present in protected form, then the N-terminal protecting group is removed, e.g. in the case of tert-butoxycarbonylamino with hydrogen chloride in dioxane.

Pharmaceutical Preparations and Uses:

The invention relates in particular to a method of treating warm-blooded animals, especially humans, suffering from a disease mentioned above, especially a proliferative disease, especially a tumor disease and in particular such a disease which responds to inhibition of the multicatalytic proteasome complex, which method comprises administering, to warm-blooded animals requiring such treatment, an amount of a compound of formula I that is effective in inhibiting tumors, to the use of a compound of formula I for such treatment, or to the use of a compound of formula I for the preparation of a pharmaceutical composition for such treatment. The invention relates also to the use of a compound of formula I in the inhibition of the multicatalytic proteasome complex in warm-blooded animals, in particular humans.

Effective doses, for example daily doses of approximately 0.05 g to about 10 g, preferably about 0.1 g to about 5 g, for example about 0.15 g to 1.5 g, of a compound of formula I are administered to a warm-blooded animal of approximately 70 kg body weight according to species, age, individual condition, mode of administration and the individual syndrome.

The invention relates also to pharmaceutical compositions comprising a compound of the formula I or a pharmaceutically acceptable salt thereof (active ingredient), especially an effective amount, most especially an amount effective in the prevention or therapy of one of the above-mentioned diseases, of the active ingredient together with pharmaceutically acceptable carriers that are suitable for topical, enteral, for example oral or rectal, or parenteral administration, and may be inorganic or organic, solid or liquid, or any combination thereof. For oral administration there are used especially tablets or gelatin capsules comprising the active ingredient together with diluents, for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerol, and/or lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Tablets may also comprise binders, for example magnesium aluminium silicate, starches, such as corn, wheat or rice starch, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and, if desired, disintegrators, for example starches, agar, alginic acid or a salt thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, dyes, flavourings and sweeteners. The pharmacologically active compounds of the present invention can also be used in the form of parenterally administrable compositions or in the form of infusion solutions. Such solutions are preferably isotonic aqueous solutions or suspensions, which, for example in the case of lyophilised compositions that comprise the active ingredient alone or together with a carrier, for example mannitol, can be prepared before use. The pharmaceutical compositions may be sterilised and/or may comprise excipients, for example preservatives, stabilisers, wetting agents and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers. The present pharmaceutical compositions which, if desired, may comprise further pharmacologically active substances, such as antibiotics, are prepared in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising processes. The concentrations of active ingredient(s) will, of course, vary depending e.g. on the compound of formula I employed, the treatment desired and the nature of the form. The present pharmaceutical compositions comprise approximately from 1% to 100%, especially from approximately 1% to approximately 20%, active ingredient(s).

Compositions for oral administration can for example be obtained by formulating a compound of formula I with a carrier medium comprising a hydrophilic phase, a lipophilic phase and a surfactant. Preferably the composition is in the form of a “microemulsion preconcentrate” or “emulsion preconcentrate”, in particular of the type providing o/w (oil-in-water) microemulsions or emulsions. However, the composition may be in the form of a microemulsion or an emulsion which additionally contains an aqueous phase, preferably water. A “microemulsion preconcentrate” is a formulation which spontaneously forms a microemulsion in an aqueous medium, for example in water or in the gastric juices after oral application. A microemulsion is a non-opaque or substantially non-opaque colloidal dispersion that is formed spontaneously or substantially spontaneously when its components are brought into contact. A microemulsion is thermodynamically stable. An “emulsion preconcentrate” is a formulation which spontaneously forms an emulsion in an aqueous medium, for example in water or in the gastric juices, after oral application. The emulsion formed is, opaque and thermodynamically stable. The lipophilic phase may comprise about 10 to 85% by weight of the carrier medium; the surfactant may comprise about 5 to 80% by weight of the carrier medium; and the hydrophilic phase may comprise about 10 to 50% by weight of the carrier medium.

The hydrophilic phase may be selected from e.g. Transcutol® (C₂H₅—[O—(CH₂)₂]₂—OH), Glycofurol® (also known as tetrahydrofurfuryl alcohol polyethylene glycol ether) and 1,2-propylene glycol, or mixtures thereof, and is preferably 1,2-propylene glycol. It may include further hydrophilic co-components, for example C₁-C₅alkanols.

Preferred lipophilic phase components are medium chain fatty acid triglycerides, mixed mono-, di-, tri-glycerides, and transesterified ethoxylated vegetable oils.

Examples of Suitable Surfactants Are:

-   -   i) reaction products of a natural or hydrogenated castor oil and         ethylene oxide. The oils available under the trade name         Cremophor® (BASF AG, Germany) are especially suitable.         Particularly suitable are Cremophor RH 40® (resulting from a         1:40 to 1:45 ratio of hydrogenated castor oil to ethylene oxide         during synthesis) and Cremophor RH 60® (resulting from a 1:60         ratio of hydrogenated castor oil to ethylene oxide during         synthesis). Also suitable are polyethyleneglycol castor oils         such as that available under the trade name Cremophor EL®         (resulting from a 1:35 to 1:40 ratio of castor oil to ethylene         oxide during synthesis). Similar or identical products which may         also be used are available under the trade names Nikkol®,         Mapeg®, Incrocas® (Croda Inc., USA) and Tagat® (Th. Goldschmidt,         Germany);     -   ii) polyoxyethylene-sorbitan-fatty acid esters, for example         esters of the type known and commercially available under         thetrade name Tween® (ICI Americas, Inc., USA);     -   iii) polyoxyethylene fatty acid esters, for example         polyoxyethylene stearic acid esters of the type known and         commercially available under the trade name Myrj® (ICI Americas,         Inc., USA).     -   iv) polyoxyethylene-polyoxypropylene co-polymers and block         copolymers, e.g. of the type known and commercially available         under the trade names Pluronic® (BASF AG, Germany), Emkalyx® and         Poloxamer®, especially preferred are Pluronic F68® and Poloxamer         188®;     -   v) Dioctylsulfosuccinate or di-[2-ethylhexyl]-succinate.     -   vi) Phospholipids, in particular lecithins.     -   vii) Propylene glycol mono- and di-fatty acid esters.

The surfactant selected preferably has an HLB (hydrophilic/lipophilic balance) of at least 10. Full physical characteristics of the products referred to herein by trade name can be obtained e.g. from H. P. Fiedler, “Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und Angrenzende Gebiete”, Editio Cantor, D-7960 Aulendorf, Germany, 3rd revised and expanded edition (1989).

For example, a suitable pharmaceutical preparation is formulated from a Cremophor®/ethanol 2:1 mixture which, for i.v. injection purposes, is diluted 1:4 (v/v) with a 5% dextrose solution in water.

The pharmaceutical compositions may also include further additives or ingredients, for example antioxidants. They exhibit especially advantageous properties when administered orally, for example in terms of consistency and high level of bioavailability obtained in standard bioavailability trials. Pharmacokinetic parameters, for example absorption and blood levels, also become surprisingly more predictable and problems in administration with erratic absorption may be eliminate or reduced. Additionally, the pharmaceutical composition is effective with tenside materials, for example bile salts, present in the gastro-intestinal tract.

The pharmaceutical compositions for oral use are preferably compounded in unit dosage form, for example by filling them into orally administrable capsule shells. The capsule shells may be soft or hard gelatine capsule shells. However, if desired the pharmaceutical compositions may be in a drink solution form and may include water or any other aqueous system, to provide emulsion or microemulsion systems suitable for drinking.

The present invention relates in particular to the use of compounds of formula I in the manufacture of a pharmaceutical composition for the treatment of a proliferative disease, e.g. of a solid tumor, and their use for the treatment of such a proliferative disease.

EXAMPLES

The following Examples illustrate the invention but do not limit the scope thereof in any way.

Abbreviations: abs. absolute i-BuB(OH)₂ Isobutyl-boronic acid DIEA N-Ethyldiisopropylamine DMF N,N-Dimethyl-formamide equiv equivalent(s) ES-MS Electrospray Mass Spectroscopy EtOAc ethyl acetate h hour(s) HPLC High Performance Liquid Chromatography MeOH methanol min minute(s) m.p. melting point MPLC Medium Pressure Liquid Chromatography R_(f) ratio of fronts value obtained by TLC on silica gel 60 F254 (Merck, Darmstadt) rt room temperature TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate TFA trifluoroacetic acid TLC Thin Layer Chromatography t_(R) retention time

Ratios of eluents and other solvent mixtures are given in volume by volume (v/v), if not mentioned otherwise.

Visualization of TLC:

TLC spots of final compounds or interemediates that are not detectable by UV-irradiation are visualized using a potassium permanganate staining solution followed by heating the plate. Composition of potassium permanganate staining solution:

-   -   2.5 g of KMnO₄ (Potassium permanganate) (Fluka, Buchs,         Switzerland) in 800 ml of H₂O and 200 ml of 1N H₂SO₄

Analytical HPLC Conditions:

System 1

Linear gradient 2-100% CH₃CN (0.1% TFA) and H₂O (0.1% TFA) in 10 min+2 min 100% CH₃CN (0.1% TFA); detection at 215 nm, flow rate 0.7 mL/min at 25° C. Column: Nucleosil 120-3 C18 (125×3.0 mm).

System 2

Linear gradient 20-100% CH₃CN (0.1% TFA) and H₂O (0.1% TFA) in 7 min+2 min 100% CH₃CN (0.1% TFA); detection at 215 nm, flow rate 1 mL/min at 30° C. Column: Nucleosil 100-3 C8HD (125×4 mm).

System 3

Linear gradient 20-100% CH₃CN (0.1% TFA) and H₂O (0.1% TFA) in 7 min+2 min 100% CH₃CN (0.1% TFA); detection at 215 nm, flow rate 1 mL/min at 30° C. Column: Nucleosil 100-3 C8 HD (125×4 mm).

Example 1 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

Step A: A 4N solution of HCl in dioxane (5.7 mL, 22.77 mMol, 30 equiv) is added to a cold (0° C.) solution of {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester ((A) in Synthetic Scheme 1) (0.352 g, 0.759 mMol) in CH₂Cl₂ abs. (5.5 mL), under an argon atmosphere. The resulting mixture is allowed to warm to rt and stirred for 10 min. Additional 4N HCl (1.9 mL, 7.59 mMol, 10 equiv) is added. The reaction mixture is stirred for 10 min and concentrated to afford the crude hydrochloride as a yellow foam.

Step B: DIEA abs. (0.72 mL, 4.14 mMol, 5 equiv) is added dropwise (1.9 mL/min) to a cold (0° C.) solution of the crude hydrochloride (0.331 g, 0.828 mMol), (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid ((B) in Synthetic Scheme 1) (0.518 g, 0.994 mMol, 1.2 equiv), and TBTU (0.292 g, 0.910 mMol, 1.1 equiv) in DMF abs. (3.0 mL), under an argon atmosphere. The reaction mixture is allowed to warm to rt, stirred for 40 min and poured onto 0° C. H₂O (45 mL). The resulting precipitate is collected by vacuum filtration, dissolved in EtOAc and washed with H₂O. The organic phase is dried (Na₂SO₄), filtered and concentrated. The residue is purified by silica gel (25 g) column chromatography (CH₂Cl₂/MeOH, 90/10) to afford the title compound as a yellow foam.

Title compound: ES-MS: 754.2 [M+H]⁺; HPLC: single peak at t_(R)=11.85 min (System 1); R_(f)=0.72 (CH₂Cl₂/MeOH, 90/10).

The starting materials are prepared as follows:

(a) (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (for Step B)

The title compound is prepared by heating a suspension of 3-bromo-biphenyl (1.47 mL, 8.54 mMol, Aldrich 25,538-6), (S)-2-amino-3-(3,4,5-trimethoxy-phenyl)-propionic acid (3,4,5-OCH₃-phe-OH) (3.27 g, 12.81 mMol), K₂CO₃ (1.18 g, 8.54 mMol) and Cul (163 mg, 0.854 mMol) in DMF abs. (10.6 mL) for 24 h at 90° C., under an argon atmosphere. The resulting mixture is allowed to cool to rt, then concentrated in vacuo and purified by MPLC (CH₃CN/H₂O/TFA) to afford the title compound.

Title compound: ES-MS: 408.2 [M+H]⁺; HPLC: single peak at t_(R)=8.86 min (System 1).

(b) (S)-2-Amino-3-(3,4,5-trimethoxy-phenyl)-propionic acid (L-3,4,5-Trimethoxy-phenylalanine)

The title compound is preprared from commercially available 3,4,5-trimethoxybenzaldehyde and N-acetylglycine according to a literature procedure (E. M. Oltz, R. C. Bruening, M. J. Smith, K. Kustin and K. Nakanishi in J. Am. Chem. 1998, 110 (18), 6162-6172). The resolution of the racemic N-acetyl-3,4,5-trimethoxy-phenylalanine methyl ester is performed by enzyme-catalyzed hydrolysis of the L-ester using Alcalase® (Novo Nordisk) as described in the literature (J. J. Nestor, Jr., T. L. Ho, R. A. Simpson, B. L. Homer, G. H. Jones, G. I. McRae and B. H. Vickery in J. Med. Chem. 1982, 25 (7), 795-801; or O. D. Tyagi & P. M. Boll in Indian J. Chem. 1992, pp. 851-854).

Title compound: [α]_(D) ²⁰ =−18.9° (c=1.025, H₂O); ES-MS: 256.1 [M+H]⁺; HPLC: single peak at t_(R)=2.08 min (System 2).

(c) {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester

The title compound is prepared as described in step B of example 1 but using (S)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylammonium trifluoroacetate ((C) in Synthetic Scheme 1) (for preparation see Kettner, C. A. and Shenvi, A. B. J. Biol. Chem. 1984, 259, p. 15106-15114 and Matteson, D. S. and Sadhu, K. M. J. Am. Chem. Soc. 1981, 103, p. 5241-5242) (2.395 g, 6.32 mMol), Boc-L-valine (1.373 g, 6.32 mMol), TBTU (2.23 g, 6.95 mMol, 1.1 equiv), DIEA (3.3 mL, 18.95 mMol, 3.0 equiv) and DMF (24 mL).

Title compound: ES-MS: 465.1 [M+H]⁺; HPLC: single peak at t_(R)=9.95 min (System 1).

Example 2 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

i-BuB(OH)₂ is added to a mixture of (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-N-[(R-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide, methanol (4.3 mL), hexane (4.3 mL) and 1N HCl (1.45 mL). The reaction mixture is stirred for 2 h at rt and then diluted with methanol (8 mL) and hexane (8 mL). The two layers are separated. The methanol layer is washed twice with hexane, diluted with CH₂Cl₂, washed with H₂O, dried (Na₂SO₄), filtered and concentrated. The residue is dissolved in CH₂Cl₂ and purified by silica gel (20 g) column chromatography (CH₂Cl/MeOH, 80/20) to afford the title compound as a pale yellow foam.

Title compound: ES-MS: 618.2 [M−H]⁻; R_(f)=0.03 (CH₂Cl₂/MeOH, 95/5).

Example 3 (S)-3-Methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-2-[(S)-2-[2-(3-phenoxy-phenyl)-acetylamino]-3-(2,3,4-trimethoxy-phenyl)-propionylamino]-butyramide

The title compound is prepared from {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester ((A) in Synthetic Scheme 2) by reiteration of the 2-step (deprotection/coupling) procedure described in example 1 but using (S)-2-tert-Butoxycarbonylamino-3-(2,3,4-trimethoxy-phenyl)-propionic acid ((D) in Synthetic Scheme 2) and (3-Phenoxy-phenyl)-acetic acid ((F) in Synthetic Scheme 2) (Trans World Chemicals, Inc.; Rockville, Md., USA) as the partners in each coupling reaction (step B, example 1), respectively. The title compound is obtained as a white solid.

Title compound: ES-MS: 812.1 [M+H]⁺; HPLC: single peak at t_(R)=11.13 min (System 1); R_(f)=0.41 (CH₂Cl₂/MeOH, 95/5).

Step 3.1: (S)-2-Amino-3-(2,3,4-trimethoxy-phenyl)-propionic acid (L-2,3,4-Trimethoxy-phenyl-alanine)

The title compound is prepared as described for (S)-2-Amino-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1).

Title compound: ES-MS: 256.1 [M+H]⁺; HPLC: t_(R)=2.54 min (System 2); [α]_(D) ²⁰=−18.5°(c=0.99, H₂O).

Step 3.2: (S)-2-tert-Butoxycarbonylamino-3-(2,3,4-trimethoxy-phenyl)-propionic acid

The title compound is synthesised starting from (S)-2-Amino-3-(2,3,4-trimethoxy-phenyl)-propionic acid according to a procedure known in the art (M. Bodanszky in Principles of Peptide Synthesis, Akad.-Verlag, 1984).

Title compound: ES-MS: 356.1 [M+H]⁺; HPLC: t_(R)=5.35 min (System 1); [α]_(D) ²⁰=−2.5° (c=0.985, MeOH).

Example 4 (R)-3-Methyl-1-{(S)-3-methyl-2-[(S-2-[2-(3-phenoxy-phenyl)-acetylamino]-3-(2.3.4-trimethoxy-phenyl)-propionylamino]-butyrylamino}-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 676.0 [M−H]⁻; R_(f)=0.025 (CH₂Cl₂/MeOH, 95/5).

Example 5 (S)-3-Methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-2-[(S)-2-(3-phenyl-propionyl-amino)-3-(2,3,4-trimethoxy-phenyl)-propionylamino]-butyramide

The title compound is prepared from {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester by reiteration of the 2-step (deprotection/coupling) procedure described in example 1 but using (S)-2-(tert-butyloxycarbonyl-amino)-3-(2,3,4-trimethoxy-phenyl)-propionic acid and 3-Phenyl-propionic acid (Fluka, Buchs, Switzerland) as the partners in each coupling reaction (step B, example 1), respectively.

Title compound: ES-MS: 734.1 [M+H]⁺; HPLC: single peak at t_(R)=11.25 min (System 1); R_(f)=0.41 (CH₂Cl₂/MeOH, 95/5).

Example 6 (R)-3-Methyl-1-{(S)-3-methyl-2-[(S)-2-(3-phenyl-propionylamino)-3-(2,3,4-trimethoxy-phenyl)-propionylamino]-butyrylamino}-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 598.2 [M−H]⁻; R_(f)=0.025 (CH₂Cl₂/MeOH, 95/5).

Example 7 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using (S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionic acid.

Title compound: ES-MS: 694.4 [M+H]⁺; HPLC: single peak at t_(R)=12.01 min (System 1); R_(f)=0.56 (CH₂Cl₂/MeOH, 95/5).

Step 7.1: (S-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionic acid

The title compound is prepared as described for (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1) but using O-methyl-L-tyrosine (Bachem). Purification by MPLC (CH₃CN/H₂O/TFA) afforded the title compound; ES-MS: 348.3 [M+H]⁺; HPLC: single peak at t_(R)=9.52 min (System 1).

Example 8 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 558.0 [M−H]⁻; HPLC: t_(R)=6.47 min (System 3); R_(f)=0.086 (CH₂Cl₂/MeOH, 95/5).

Example 9 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using (S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 724.4 [M+H]⁺; HPLC: single peak at t_(R)=11.75 min (System 1); R_(f)=0.41 (CH₂Cl₂/MeOH, 95/5).

Step 9.1: (S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionic acid

The title compound is prepared as described for (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1) but using 3-(3,4-dimethoxyphenyl)-L-alanine (Aldrich). Purification by MPLC (CH₃CN/H₂O/TFA) afforded the title compound; ES-MS: 378.2 [M+H]⁺; HPLC: single peak at t_(R)=9.10 min (System 1).

Example 10 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 588.2 [M−H]⁻; R_(f)=0.090 (CH₂Cl₂/MeOH, 95/5).

Example 11 (S)-2-[(S)-2-(3-Isopropyl-phenylamino)-3-(3,4,5-trimethoxy-phenyl-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using (S)-2-(3-Isopropyl-phenylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 720.4 [M+H]⁺; HPLC: single peak at t_(R)=11.85 min (System 1); R_(f)=0.43 (CH₂Cl₂/MeOH, 95/5).

Step 11.1: (S-2-(3-Isopropyl-phenylamino-3-(3,4,5-trimethoxy-phenyl)-propionic acid

The title compound is prepared as described for (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1) but using 1-bromo-3-isopropylbenzene (Lancaster). Purification by MPLC (CH₃CN/H₂O/TFA) afforded the title compound; ES-MS: 374.1 [M+H]⁺; HPLC: single peak at t_(R)=8.95 min (System 1).

Example 12 (R)-1-{(S)-2-[(S)-2-(3-Isopropyl-phenylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 584.3 [M−H]⁻; R_(f)=0.13 (CH₂Cl₂/MeOH, 95/5).

Example 13 (S)-3-Methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3.5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-2-[(S)-2-(3-pyridin-2-yl-phenylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-butyramide

The title compound is prepared as described in example 1 but using (S)-2-(3-Pyridin-2-yl-phenylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 755.3 [M+H]⁺; HPLC: single peak at t_(R)=9.97 min (System 1); R_(f)=0.23 (CH₂Cl₂/MeOH, 95/5).

Step 13.1: 2-(3-Bromo-phenyl)-pyridine

The title compound is prepared according to literature procedures: Zhang, Biliang, Breslow, Ronald Ester Hydrolysis by a Catalytic Cyclodextrin Dimer Enzyme Mimic with a Metallobipyridyl Linking Group. J. Am. Chem. Soc. (1997), 119(7), 1676-1681; M. Van der Sluis, V. Beverwijk, A. Termaten, F. Bickelhaupt, H. Kooijman, A. L. Spek Synthesis of Novel Phosphaalkene-Based Bidentate Ligands Mes*P:CH(3-R—Ar) (R=Pyridyl, Carbaldimino) and Formation of Three-Membered Palladacycles Mes*(Me)P—CH(3-R—Ar)—PdCl by Carbopalladation of the P:C Double Bond. Organometallics (1999), 18(8), 1402-1407.

Title compound: ES-MS: 235.0 [M+H]⁺; HPLC: single peak at t_(R)=6.64 min (System 1); R_(f)=0.17 (Hexane/Et₂O, 80/20).

Step 13.2: (S)-2-(3-Pyridin-2-yl-phenylamino)-3-(3,4,5trimethoxy-phenyl)-propionic acid

The title compound is prepared as described for (S-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1) but using 2-(3-Bromo-phenyl)-pyridine. Purification by MPLC (CH₃CN/H₂O/TFA) afforded the title compound; ES-MS: 409.2 [M+H]⁺; HPLC: single peak at t_(R)=6.64 min (System 1).

Example 14 (R)-3-Methyl-1-{(S)-3-methyl-2-[(S)-2-(3-pyridin-2-yl-phenylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-butyrylamino}-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 619.2 [M−H]⁻; R_(f)=0.044 (CH₂Cl₂/MeOH, 95/5).

Example 15 (S)-2-[(S-2-(Biphenyl-3-ylamino)-3-(2,3,4-trimethoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3.5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using (S)-2-(Biphenyl-3-ylamino)-3-(2,3,4-trimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 754.4 [M+H]⁺; HPLC: single peak at t_(R)=12.08 min (System 1); R_(f)=0.66 (CH₂Cl₂/MeOH, 95/5).

Step 15.1: (S)-2-(Biphenyl-3-ylamino)-3-(2,3,4-trimethoxy-phenyl)-propionic acid

The title compound is prepared as described for (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1) but using (S)-2-Amino-3-(2,3,4-trimethoxy-phenyl)-propionic acid. Purification by MPLC (CH₃CN/H₂O/TFA) afforded the title compound; ES-MS: 408.2 [M+H]⁺; HPLC: single peak at t_(R)=9.42 min (System 1).

Example 16 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(2,3,4-trimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 618.3 [M−H]⁻; R_(f)=0.23 (CH₂Cl₂/MeOH, 95/5).

Example 17 (S)-2-[(S-3-(4-Benzyloxy-phenyl)-2-(biphenyl-3-ylamino)-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using (S)-3-(4-Benzyloxy-phenyl)-2-(biphenyl-3-ylamino)-propionic acid.

Title compound: ES-MS: 770.3 [M+H]⁺; HPLC: single peak at t_(R)=12.45 min (System 1); R_(f)=0.74 (CH₂Cl₂/MeOH, 95/5).

Step 17.1: (S)-3-(4-Benzyloxy-phenyl)-2-(biphenyl-3-ylamino)-propionic acid

The title compound is prepared as described for (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1) but using (S)-2-Amino-3-(4benzyloxy-phenyl)-propionic acid (O-benzyl-L-tyrosine). Purification by MPLC (CH₃CN/H₂O/TFA) afforded the title compound; ES-MS: 424.3 [M+H]⁺; HPLC: single peak at t_(R)=10.40 min (System 1).

Example 18 (R)-1-{(S)-2-[(S)-3-(4-Benzyloxy-phenyl)-2-(biphenyl-3-ylamino)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 633.9 [M−H]⁻; R_(f)=0.65 (CH₂Cl₂/MeOH, 90/10).

Example 19 (R)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using {(R)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester.

Title compound: ES-MS: 754.1 [M+H]⁺; HPLC: single peak at t_(R)=11.73 min (System 1); R_(f)=0.52 (CH₂Cl₂/MeOH, 95/5).

Step 19.1: {(R)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester

The title compound is prepared as described for {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester (example 1 (c)) but using Boc-D-valine (Fluka).

Title compound: ES-MS: 465.4 [M+H]⁺.

Example 20 (R)-1-{(R)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 618.2 [M−H]⁻; R_(f)=0.088 (CH₂Cl₂/MeOH, 95/5).

Example 21 (S)-2-[(R)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using (R)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 754.3 [M+H]⁺; HPLC: single peak at t_(R)=11.71 min (System 1); R_(f)=0.67 (CH₂Cl₂/MeOH, 95/5).

Step 21.1: (R)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid

The title compound is prepared as described for (S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid (example 1) but using (R)-2-amino-3-(3,4,5-trimethoxy-phenyl)-propionic acid (3,4,5-OCH₃-phe-OH).

Title compound: ES-MS: 408.2 [M+H]⁺; HPLC: single peak at t_(R)=9.10 min (System 1).

For the synthesis of (R)-2-amino-3-(3,4,5-trimethoxy-phenyl)-propionic acid see example 1. After the enzymatic resolution, the remaining D-aminoacid-methylester is hydrolysed and deacetylated using protocols known in the art; [α]_(D) ²=+19.7° (c=1.04, H₂O); ES-MS: 256.2 [M+H]⁺; single peak at t_(R)=2.11 min (System 2).

Example 22 (R)-1-{(S)-2-[(R)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 618.2 [M−H]⁻; R_(f)=0.20 (CH₂Cl₂/MeOH, 90/10).

Example 23 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-N-[3-methyl-1-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-butyl]-butyramide

The title compound is prepared as described in example 1 but using {(S)-2-Methyl-1-[3-methyl-1-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester.

The title compound is obtained as a crude product, ES-MS: 702.3 (M+H]⁺; HPLC: t_(R)=10.31 min (System 1).

Step 23.1: {(S-2-Methyl-1-[3-methyl-1-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester

The title compound is prepared in analogy to the synthesis of {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester (example 1 (c)).

Title compound: ES-MS: 413.3 [M+H]⁺.

Example 24 1-{(S)-2-[(S-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 618.2 [M−H]⁻; R_(f)=0.076 (CH₂Cl₂/MeOH, 95/5); HPLC: two peaks at t_(R)=6.23 min and 6.36 min (ratio 1:1) (System 3).

Example 25 (S)-2-{(S)-3-(3,4-Dimethoxy-phenyl)-2-[2-(3-phenoxy-phenyl)-acetylamino]-propionylamino}-3-methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared from {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester by reiteration of the 2-step (deprotection/coupling) procedure described in example 1 but using Boc-L-3,4-dimethoxyphenylalanine (Synthetech) and (3-Phenoxy-phenyl)-acetic acid (Trans World Chemicals, Inc.; Rockville, Md., USA) as the partners in each coupling reaction (step B, example 1), respectively. The title compound is obtained as a foam; ES-MS: 782.3 [M+H]⁺; HPLC: single peak at t_(R)=11.76 min (System 1); R_(f)=0.61 (CH₂Cl₂/MeOH, 90/10).

Example 26 (R)-1-((S)-2-{(S)-3-(3,4-Dimethoxy-phenyl)-2-[2-(3-phenoxy-phenyl)-acetylamino]-propionylamino}-3-methyl-butyrylamino)-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 646.2 [M−H]⁻; HPLC: single peak at t_(R)=5.90 min (System 3); R_(f)=0.12 (CH₂Cl₂/MeOH, 90/10).

Example 27 (S)-3-Methyl-N-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-2-[(S)-2-[2-(3-phenoxy-phenyl)-acetylamino]-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-butyramide

The title compound is prepared from {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester by reiteration of the 2-step (deprotection/coupling) procedure described in example 1 but using (S-2-tert-Butoxycarbonylamino-3-(3,4,5-trimethoxy-phenyl)-propionic acid and (3-Phenoxy-phenyl)-acetic acid (Trans World Chemicals, Inc.; Rockville, Md., USA) as the partners in each coupling reaction (step B, example 1), respectively. The title compound is obtained as a yellow foam; ES-MS: 812.4 [M+H]⁺; HPLC: single peak at t_(R)=11.36 min (System 1); R_(f)=0.53 (CH₂Cl₂/MeOH, 95/5).

Step 27.1: (S-2-tert-Butoxycarbonylamino-3-(3,4,5-trimethoxy-phenyl)-propionic acid

The title compound is synthesised as described for (S)-2-tert-Butoxycarbonylamino-3-(2,3,4-trimethoxy-phenyl)-propionic acid (Example 3) but starting from (S)-2-Amino-3-(3,4,5-trimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 356 [M+H]⁺; HPLC: t_(R)=4.83 min (System 2); m.p.=76-80° C.; [α]_(D) ²⁰=+13.4° (c=1.01, methanol).

Example 28 (R)-3-Methyl-1-{(S)-3-methyl-2-[(S)-2-[2-(3-phenoxy-phenyl)-acetylamino]-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-butyrylamino}-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 676.2 [M−H]⁻; R_(f)=0.14 (CH₂Cl₂/MeOH, 95/5).

Example 29 (S)-2-{(S)-3-(4-Benzyloxy-phenyl)-2-[2-(3-benzyloxy-phenyl)-acetylamino]-propionylamino}-3-methyl-N-[(R-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-butyramide

The title compound is prepared from {(S)-2-Methyl-1-[(R-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester by reiteration of the 2-step (deprotection/coupling) procedure described in example 1 but using (S)-3-(4-Benzyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acid and (3-Phenoxy-phenyl)-acetic acid (Trans World Chemicals, Inc.; Rockville, Md., USA) as the partners in each coupling reaction (step B, example 1), respectively. The title compound is obtained as a beige foam; ES-MS: 842.0 [M+H]⁺; HPLC: single peak at t_(R)=12.19 min (System 1); R_(f)=0.37 (CH₂Cl₂/MeOH, 95/5).

Step 29.1: (S)-3-(4-Benzyloxy-phenyl)-2-tert-butoxycarbonylamino-propionic acid

The title compound is synthesised as described for (S-2-tert-Butoxycarbonylamino-3-(2,3,4-trimethoxy-phenyl)-propionic acid (Example 3) but starting from O-Benzyl-L-tyrosine (Fluka).

Title compound: ES-MS: 370.1 [M−H]⁻; HPLC: t_(R)=9.23 min (System 1).

Example 30 (R)-1-((S)-2-{(S)-3-(4-Benzyloxy-phenyl)-2-[2-(3-benzyloxy-phenyl)-acetylamino]-propionylamino}-3-methyl-butyrylamino)-3-methyl-butylboronic acid

The titled compound is prepared as described in example 2; ES-MS: 705.8 [M−H]⁻; R_(f)=0.12 (CH₂Cl₂/MeOH, 95/5).

Example 31 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-4-methyl-pentanoic acid [(R-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-amide

The title compound is prepared as described in example 1 but using {(S)-3-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-butyl}-carbamic acid tert-butyl ester.

Title compound: ES-MS: 768.2 [M+H]⁺; HPLC: single peak at t_(R)=11.79 min (System 1); R_(f)=0.72 (CH₂Cl₂/MeOH, 95/5).

Step 31.1: {(S)-3-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-butyl}-carbamic acid tert-butyl ester

The title-compound is prepared as described for {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcamoyl]-propyl}-carbamic acid tert-butyl ester (example 1 (c)) but using Boc-L-leucine.

Title compound: ES-MS: 479.2 [M+H]⁺; HPLC: single peak at t_(R)=10.05 min (System 1).

Example 32 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-4-methyl-pentanoylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 632.2 [M−H]⁻; R_(f)=0.15 (CH₂Cl₂/MeOH, 95/5).

Example 33 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionylamino]-4-methyl-pentanoic acid [(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-amide

The title compound is prepared as described in example 1 but using {(S)-3-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-butyl}-carbamic acid tert-butyl ester and (S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 738.3 [M+H]⁺; HPLC: single peak at t_(R)=11.76 min (System 1); R_(f)=0.59 (CH₂Cl₂/MeOH, 95/5).

Example 34 (R)-1-{(S-2-[(S)-2-(Biphenyl-3-ylamino-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-4-methyl-pentanoylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 602.2 [M−H]⁻; R_(f)=0.14 (CH₂C₂/MeOH, 95/5).

Example 35 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionylamino]-4-methyl-pentanoic acid [(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butyl]-amide

The title compound is prepared as described in example 1 but using {(S)-3-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-butyl}-carbamic acid tert-butyl ester and (S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionic acid.

Title compound: ES-MS: 708.3 [M+H]⁺; HPLC: single peak at t_(R)=12.03 min (System 1); R_(f)=0.70 (CH₂C₂/MeOH, 95/5).

Example 36 (R)-1-{(S-2-[(S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionylamino]-4-methyl-pentanoylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 572.1 [M−H]⁻; R_(f)=0.25 (CH₂Cl₂/MeOH, 90/10).

Example 37 (S)-2-(Biphenyl-3-ylamino)-N-{(S)-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl-ethyl}-3-(3,4,5-trimethoxy-phenyl)-propionamide

The title compound is prepared as described in example 1 but using {(S)-1-[(R)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester.

Title compound: ES-MS: 726.3 [M+H]⁺; HPLC: single peak at t_(R)=11.24 min (System 1); R_(f)=0.41 (CH₂Cl₂/MeOH, 95/5).

Step 37.1: {(S)-1-[(R-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester

The title compound is prepared as described for {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester (step 1.1, example 1) but using Boc-L-alanine (Fluka).

Title compound: ES-MS: 437.4 [M+H]⁺; HPLC: single peak at t_(R)=10.91 min (System 1).

Example 38 (R)-1-{(S)-2-[(S-2-Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-propionylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 590.0 [M−H]⁻; R_(f)=0.12 (CH₂Cl₂/MeOH, 95/5).

Example 39 (S)-2-(Biphenyl-3-ylamino)-N-{(S)-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-3-(2,3,4-trimethoxy-phenyl-propionamide

The title compound is prepared as described in example 1 but using {(S)-1-[(R)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester and (S)-2-(Biphenyl-3-ylamino)-3-(2,3,4-trimethoxy-phenyl)-propionic acid.

Title-compound: ES-MS: 726.3 [M+H]⁺; HPLC: single peak at t_(R)=11.71 min (System 1); R_(f)=0.45 (CH₂Cl₂/MeOH, 95/5).

Example 40 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(2,3,4-trimethoxy-phenyl)-propionylamino]-propionylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 590.0 [M−H]⁻; R_(f)=0.033 (CH₂Cl₂/MeOH, 95/5).

Example 41 (S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-N-{(S)-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-propionamide

The title compound is prepared as described in example 1 but using {(S)-1-[(R)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester and (S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionic acid.

Title compound: ES-MS: 666.3 [M+H]⁺; HPLC: single peak at t_(R)=11.63 min (System 1); R_(f)=0.46 (CH₂Cl₂/MeOH, 95/5).

Example 42 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino-3-(4-methoxy-phenyl)-propionylamino]-propionylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 530.3 [M−H]⁻; R_(f)=0.051 (CH₂Cl₂/MeOH, 95/5).

Example 43 (S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-N-{(S)-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4yl)-butylcarbamoyl]-ethyl}-propionamide

The title compound is prepared as described in example 1 but using {(S)-1-[(R)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester and (S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 696.3 [M+H]⁺; HPLC: single peak at t_(R)=11.39 min (System 1); R_(f)=0.53 (CH₂Cl₂/MeOH, 95/5).

Example 44 (R)-1-{(S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4-dimethoxy-phenyl)-propionylamino]-propionylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 560.2 [M−H]⁻; R_(f)=0.023 (CH₂Cl₂/MeOH, 95/5).

Example 45 (S)-2-(3-Isopropyl-phenylamino)-N-{(S)-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-3-(3,4,5-trimethoxy-phenyl)-propionamide

The title compound is prepared as described in example 1 but using {(S)-1-[(R)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester and (S)-2-(3-Isopropyl-phenylamino)-3-(3,4,5-trimethoxy-phenyl)-propionic acid.

Title compound: ES-MS: 692.3 [M+H]⁺; HPLC: single peak at t_(R)=11.49 min (System 1); R_(f)=0.24 (CH₂Cl₂/MeOH, 95/5).

Example 46 (R)-1-(S)-2-{(S)-2-(3-Isopropyl-phenylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-propionylamino}-3-methyl-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 560.2 [M−H]⁻; R_(f)=0.22 (CH₂Cl₂/MeOH, 95/5).

Example 47 (S)-N-{(S)-1-[(R)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl-butylcarbamoyl]-ethyl}-2-(3-phenyl-propionylamino)-3-(2,3,4-trimethoxy-phenyl)-propionamide

The title compound is prepared from {(S)-1-[(R)-3-Methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-ethyl}-carbamic acid tert-butyl ester by reiteration of the 2-step (deprotection/coupling) procedure described in example 1 but using (S)-2-Amino-3-(2,3,4-trimethoxy-phenyl)-propionic acid and 3-Phenyl-propionic acid (Fluka) as the partners in each coupling reaction (step B, example 1), respectively.

Title compound: ES-MS: 706.3 [M+H]⁺; HPLC: single peak at t_(R)=10.81 min (System 1); R_(f)=0.32 (CH₂Cl₂/MeOH, 95/5).

Example 48 (R)-3-Methyl-1-{(S)-2-[(S)-2-(3-phenyl-propionylamino)-3-(2,3,4-trimethoxy-phenyl)-propionylamino]-propionylamino}-butylboronic acid

The title compound is prepared as described in example 2; ES-MS: 570.3 [M−H]⁻; R_(f)=0.22 (CH₂Cl₂/MeOH, 95/5).

Example 49 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-2-phenyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-ethyl]-butyramide

The title compound is prepared as described in example 1 but using {(S)-2-Methyl-1-[(R)-2-phenyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-ethylcarbamoyl]-propyl}-carbamic acid tert-butyl ester.

Title compound: ES-MS: 788.0 [M+H]⁺; HPLC: single peak at t_(R)=11.66 min (System 1); R_(f)=0.79 (CH₂Cl₂/MeOH, 95/5).

Step 49.1: {(S)-2-Methyl-1-[(R)-2-phenyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-ethylcarbamoyl]-propyl}-carbamic acid tert-butyl ester

The title compound is prepared in analogy to the synthesis of {(S)-2-Methyl-1-[(R)-3-methyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-butylcarbamoyl]-propyl}-carbamic acid tert-butyl ester (example 1 (c)).

Title compound: ES-MS: 499.1 [M+H]⁺; HPLC: single peak at t_(R)=10.78 min (System 1).

Example 50 (R)-1-{(S)-2-[(S-2-(Biphenyl-3-ylamino)-3-(3,4,5-trimethoxy-phenyl)-propionylamino]-3-methyl-butyrylamino}-2-phenyl-ethylboronic acid

The title compound is prepared as described in example 2; ES-MS: 652.2 [M−H]⁻; R_(f)=0.22 (CH₂C₂/MeOH, 95/5).

Example 51 (S)-2-[(S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionylamino]-3-methyl-N-[(R)-2-phenyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl-ethyl]-butyramide

The title compound is prepared as described in example 1 but using {(S)-2-Methyl-1-[(R)-2-phenyl-1-((1S,2S,6R,8S)-2,9,9-trimethyl-3,5-dioxa-4-bora-tricyclo[6.1.1.0^(2,6)]dec-4-yl)-ethylcarbamoyl]-propyl}-carbamic acid tert-butyl ester and (S)-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionic acid.

Title compound: ES-MS: 727.9 [M+H]⁺; HPLC: single peak at t_(R)=11.87 min (System 1); R_(f)=0.73 (CH₂Cl₂/MeOH, 95/5).

Example 52 (R)-1-{(S)-2-[(S-2-(Biphenyl-3-ylamino)-3-(4-methoxy-phenyl)-propionylamino]-3methyl-butyrylamino}-2-phenyl-ethylboronic acid

The title compound is prepared as described in example 2; ES-MS: 591.8 [M−H⁻]; R_(f)=0.13 (CH₂Cl₂/MeOH, 95/5).

Example 53 Inhibition of the chymotrypsin-like activity of the 20S proteasome

Exemplary IC₅₀ values determined according to the test described above for compounds of formula I are given below (Table 1). TABLE 1 IC₅₀ [μM] (results of one Example or two experiments) 1 0.0046/0.0024 2 0.0028/0.0021 3 0.0017/0.0014 4 0.0019/0.0015 5 0.0013/0.0006 6 0.0018/0.0019 7 0.0029/0.0032 8 0.0028/0.0045 9 0.0017/0.0022 10 0.0029/0.004  11 0.0039 12 0.0038 13 0.0013 14 0.0017 15 0.0071 16 0.0059 17 0.0093 18 0.0015 21 0.0015 22 0.0017 23 0.0021 24 0.0021 25 0.0008 26 0.001 27 0.0003 28 0.0008 29 0.004 30 0.0059 31 0.0022 32 0.0037 33 0.0026 34 0.0013 35 0.0023 36 0.0023 37 0.0013 38 0.0017 39 0.0019 40 0.0022 41 0.0012 42 0.0019 43 0.0018 44 0.001 45 0.0013 46 0.0019 47 0.0008 48 0.0007 50 0.0023 51 0.0043 52 0.005

Example 54 Composition for Oral Application

20.0 g of a solution for oral application of any one of the title compounds given in Examples 1 to 6 can be prepared as follows (% means weight ingredient/total weight solution): Cremophor RH 40 ® 9.6 g (48%), corn-oil-mono-di-tri-glycerides 5.8 g (29%), propylene glycol 3.8 g (19%), compound of formula I 0.8 g (4%).

The solution is prepared freshly before use. 

1-20. (canceled)
 21. A pharmaceutical composition for the treatment of a proliferative disease in warm-blooded animals, including humans, comprising, in a dose effective against said disease, a compound according to formula I

wherein R₁ is unsubstituted or substituted aryl; arylalkylcarbonyl, wherein the aryl moiety is unsubstituted or substituted; unsubstituted or substituted heterocyclyl; or heterocyclylalkylcarbonyl, wherein the heterocyclyl moiety is unsubstituted or substituted; R₂ is unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl; R₃ is hydrogen, unsubstituted or substituted aryl or alkyl which is unsubstituted or substituted by unsubstituted or substituted cycloalkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl comprising at least one nitrogen atom; R₄ is a moiety of the formula IA,

wherein A₁ and A₂ are hydroxy or substituted hydroxy, or together with the binding boron atom and the two binding oxygen atoms form a ring of the formula IA*,

wherein W is alkylene, substituted alkylene, unsubstituted or substituted cycloalkylene, unsubstituted or substituted bicycloalkylene or unsubstituted or substituted tricycloalkylene; and R₅ is unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, or unsubstituted or substituted cycloalkyl; or a pharmaceutically acceptable salt of such a compound, together with a pharmaceutically acceptable carrier, wherein the proliferative disease is selected from psoriasis, restenosis, muscle protein degradation or other diseases connected with intracellular protein degradation or negative nitrogen balance, sepsis, burns, trauma, chronic or systemic infections, neuromotor degenerative disease, muscular dystrophia, acidosis, spinal or nerve injuries, diseases related to antigen presentation on cells; and diseases related to cell adhesion.
 22. A method of treating warm-blooded animals, including humans, which comprises administering to such warm-blooded animal suffering from a proliferative disease a dose, effective against said disease, of a compound according to formula I

wherein R₁ is unsubstituted or substituted aryl; arylalkylcarbonyl, wherein the aryl moiety is unsubstituted or substituted; unsubstituted or substituted heterocyclyl; or heterocyclylalkylcarbonyl, wherein the heterocyclyl moiety is unsubstituted or substituted; R₂ is unsubstituted or substituted aryl or unsubstituted or substituted heteroaryl; R₃ is hydrogen, unsubstituted or substituted aryl or alkyl which is unsubstituted or substituted by unsubstituted or substituted cycloalkyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl comprising at least one nitrogen atom; R₄ is a moiety of the formula IA,

wherein A₁ and A₂ are hydroxy or substituted hydroxy, or together with the binding boron atom and the two binding oxygen atoms form a ring of the formula IA*,

wherein W is alkylene, substituted alkylene, unsubstituted or substituted cycloalkylene, unsubstituted or substituted bicycloalkylene or unsubstituted or substituted tricycloalkylene; and R₅ is unsubstituted or substituted alkyl, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclyl, or unsubstituted or substituted cycloalkyl; or of a pharmaceutically acceptable salt of such a compound, wherein the proliferative disease is selected from psoriasis, restenosis, muscle protein degradation or other diseases connected with intracellular protein degradation or negative nitrogen balance, sepsis, burns, trauma, chronic or systemic infections, neuromotor degenerative disease, muscular dystrophia, acidosis, spinal or nerve injuries, diseases related to antigen presentation on cells; and diseases related to cell adhesion. 